Super-resolution microscope system and method for illumination

ABSTRACT

A microscope system comprising an adjusted specimen and a microscope body, wherein the adjusted specimen is dyed with molecule which has three electronic states including at least a ground state and in which an excited wavelength band from the first electron excited state to the second electron excited state overlaps a fluorescent wavelength band upon deexcitation through a fluorescence process from the first electron excited state to a vibrational level in the ground state. There is provided a novel microscope system which is enabled to condense an erase light for exciting a molecule in the first electron excited state to the second electron excited state in an excellent beam profile by using a simple, compact optical system and which has high stability and operability and an excellent super-resolution.

TECHNICAL FIELD

The invention of this application relates to a microscope system. Moreparticularly, the invention of this application relates to a novelmicroscope system of high performance and function, which is able toachieve a high quality image of a high spatial resolution byilluminating a dyed specimen with lights of plural wavelengths.

BACKGROUND ART

In the prior art, there have been developed various types of opticalmicroscopes, and their performance has been enhanced according to thedevelopment in the peripheral technique including the laser techniqueand the electronic graphic technique. As one of these high performanceoptical microscopes, there has been proposed (in Japanese PatentApplication No. 6-329165) a microscope which is able, by using a doubleresonance absorption process induced by illuminating a specimen withlights of plural wavelengths, not only to control the contrast of animage to be obtained but also to perform a chemical analysis.

This optical microscope can, by using the double resonance absorption,select a specific molecule and observe absorption and fluorescencecaused by a specific optical transition. First, an electron of a valenceorbit 2, owned by the molecule in a ground state illustrated in FIG. 1,is excited to a valence orbit 3 that is a vacant orbit by lightirradiation, as illustrated in FIG. 2. This is a first excited state.Next, the electron on a valence orbit 1 is excited, as illustrated inFIG. 3, to a hole generated on the valence orbit 2 by irradiating themwith a light of another wavelength. This is a second excited state. Themolecule then returns to the ground state while emitting fluorescence orphosphorescence, as illustrated in FIG. 4. And, an absorption image or aluminous image is observed by using the absorption process of FIG. 2 orthe emission of the fluorescence or phosphorescence of FIG. 4.

At first, when the molecule composing a specimen is to be excited to thefirst excited state with a light of a resonance wavelength λ1 by, forexample, a laser beam, the number of molecules in the first excitedstate in a unit volume increases as an intensity of the irradiationlight increases. Since a linear absorption coefficient is given as aproduct of an absorption cross-section per molecule and the number ofmolecules per unit volume, in the excitation process of FIG. 3, thelinear absorption coefficient for the light of a resonance wavelength λ2subsequently applied depends upon the intensity of the light of thewavelength λ1 applied first.

In short, the linear absorption coefficient for the wavelength λ2 can becontrolled with the intensity of the light of the wavelength λ1. Thisindicates that, when irradiating a specimen with lights of twowavelengths λ1 and λ2 and obtaining a transmission image by thewavelength λ2, contrast of the transmission image can be completelycontrolled with a quantity of the light of the wavelength λ1.

On the other hand, when the deexcitation process from the second excitedstate of FIG. 3 by fluorescence or phosphorescence is possible, theluminous intensity is proportional to the number of molecules in thefirst excited state. This makes it possible to control an imagecontrast, even when used as a fluorescent microscope.

Further, this optical microscope of the prior art is able not only tocontrol the contrast but also to perform the chemical analysis. Sincethe outermost valence orbit in FIG. 1 has an energy level intrinsic to amolecule, the wavelength λ1 is different for the molecule. At the sametime, the wavelength λ2 is also intrinsic to the molecule. As a result,the molecule to absorb or emit a light can be restricted from the twowavelengths λ1 and λ2, so that an accurate chemical composition of aspecimen can be identified.

Moreover, when the valence electron is to be excited, only a lighthaving a specific electric-field vector with respect to a molecular axisis intensively absorbed. Thus, if an absorption image or fluorescenceimage is obtained while determining the directions of polarization ofthe wavelengths λ1 and λ2, the direction of orientation can also beidentified for the same molecule.

In recent years, there has also been proposed (in Japanese PatentApplication No. 8-302232) a fluorescent microscope which has a highspatial resolution exceeding a diffraction limit by using the doubleresonance absorption process.

FIG. 5 is a conceptional diagram illustrating the double resonanceabsorption process in molecule. It is illustrated in FIG. 5 that amolecule in the ground state is excited to the first excited state withthe light of the wavelength λ1 and further to the second excited statewith the light of the wavelength λ2 and that fluorescence from thissecond excited state is extremely weak for some kinds of a molecule.

The molecule having such optical properties experiences a remarkablyinteresting phenomenon. FIG. 6 illustrates an extension of a spatialdistance in the double resonance absorption process, with an abscissabeing an X axis. In FIG. 6, there are illustrated a spatial area A1which is irradiated with the light of the wavelength λ2 and a spatialarea A0 which is not irradiated with the light of the wavelength λ2. Inthis spatial area A0, a great number of the molecules being in the firstexcited state are generated by the λ1 light excitation. At this time,fluorescence emitted with a wavelength λ3 from the spatial area A0 canbe observed. In the spatial area A1, however, the irradiation of thelight of the wavelength λ2 excites most of the molecules in the firstexcited state instantly to the second excited state at a higher level,so that the molecules in the first excited state disappears. As aresult, the fluorescence of the wavelength λ3 completely disappears, andfurther, the fluorescence from the second excited state does not existintrinsically, so that the fluorescence itself is completely inhibitedin the spatial area A1. It is therefore understood that the fluorescenceexists only in the spatial area A0.

This result has a remarkably important meaning if considered from thefield of application of the microscope. In the scannigng type lasermicroscope of the prior art, a laser beam is condensed to produce amicro beam thereby to scan a specimen to be observed. At this time, thesize of the micro beam is determined by the diffraction limit which inturn is determined by a numerical aperture of a condenser lens and awavelength, so that a higher spatial resolution cannot be expected onprinciple. However, according to FIG. 6, since the fluorescent area isinhibited with the irradiation of λ2, by overlaping the wavelengths oftwo kinds of λ1 and λ2 skillfully, the fluorescent area is made narrowerthan the size determined by the numerical aperture of the condenser lensand the wavelength, while noticing the irradiation area of λ1 forexample. Thus, the spatial resolution is substantially improved.Therefore, by adopting this principle, it is possible to provide afluorescent microscope exceeding the diffraction limit. This is asuper-resolution microscope using the double resonance absorptionprocess.

In order to enhance the super-resolution of this microscope, anotherproposal has been made (in Japanese Patent Application No. 9-25444). Amolecule of various kinds, which has three quantum states including atleast the ground state and in which a thermal relaxation process is moredominant than a relaxation process by fluorescence in transition upondeexcitation from a higher excited state excepting the first excitedstate to the ground state, is employed as a fluorescence labelermolecule. The specimen in which the fluorescence labeler molecule and abio-molecule dyed biochemically are chemically bonded, is irradiatedwith the light of the wavelength λ1 to excite the fluorescence labelermolecule to the first excited state and is then instantly excited withthe light of the wavelength λ2 to a higher quantum level, so thatfluorescence from the second excited state is inhibited, thereby toinhibit the spatial fluorescent area artificially, thus the spatialresolution can be improved.

The optical properties of such molecule can be described in thefollowing manner from the standpoint of quantum chemistry.

Generally, each atom composing a molecule is bonded by a or πbonds. Inother word, according to the quantum chemistry, molecular orbits of themolecule has an a molecular orbit or a πmolecular orbit, and an electronexisting on these molecular orbits plays an important role to bond eachatom. Of these, the electron on the a molecular orbit intensely bondseach atom to determine an inter-atomic distance in the molecule, whichis the frame of the molecule. On the contrary, the electron on the πmolecular orbit makes little contribution to the bond of each atom, butis rather bound throughout the molecule by an extremely weak force.

In most cases, if the electron on the σ molecular orbit is excited witha light, the inter-atomic distance of the molecule is highly changed tocause a drastic structure change including dissociation of the molecule.As a result, a kinetic energy of the atom and an energy given for thestructural change by the light to the molecule are mostly transformedinto a thermal energy. Because of this, an excitation energy is notconsumed in fluorescence. Further, the structural change of the moleculeoccurs at an extremely high rate, for example in time period smallerthan pico seconds, so that the fluorescence lifetime is short even iffluorescence occurs in that process. On the contrary, however, even ifthe electron on the σ molecular orbit is excited, the structure itselfof the molecule makes little change, but remains at a high quantumdiscrete level for a long time so that it is deexcited while releasingfluorescence in the order of nano seconds.

According to the quantum chemistry, the fact that a molecule has a πmolecular orbit and the fact that a molecule has a double bond areequivalent, and selection of a molecule having sufficient double bondsas a fluorescence labeler molecule is a necessary condition. Moreover,it has been confirmed (e.g., M. Fujii et. al., Chem. Phys. Lett. 171(1990) 341).that, for a six-membered ring molecule such as a benzene orpyrazine among molecules having double bonds, fluorescence from thesecond electron excited state is extremely weak. Hence, if a moleculecontaining the six-membered ring such as the benzene or pyrazine isselected as a fluorescence labeler molecule, the super-resolution of themicroscope can be effectively utilized because a fluorescence lifetimefrom the first excited state is long and because the fluorescence can beeasily inhibited by an optical excitation from the first excited stateto the second excited state.

Accordingly, if the specimen is dyed with such a fluorescence labelermolecule and is observed, not only its fluorescent image can be observedin a high spatial resolution, but also only the specific chemical groupof a bio-specimen can be selectively dyed by adjusting the chemicalgroup of the side chain of the fluorescence labeler molecule. Thus, eventhe detailed chemical composition of the specimen can be analyzed.

Generally, the double resonance absorption process occurs only when twooptical wavelengths, a polarization state and the like satisfy specificconditions, so that the molecular structure can be known in a remarkabledetail by using this fact. A polarization plane of a light and anorientation direction of a molecule have an intense correlation, so thatthe double resonance absorption process intensely occurs when eachpolarization plane of the lights of two wavelengths and the orientationdirection of the molecule make a predetermined angle. Hence, byirradiating a specimen surface simultaneously with the two wavelengthlights and by turning each polarization plane, disappearing level offluorescence is changed, so that the information on a spatialorientation of a tissue to be observed is achieved from that change.This achievement can also be made by adjusting the lights of twowavelengths.

It is understood from the description thus far made that the opticalmicroscope of the prior art using the double resonance absorptionprocess has the super-resolution and the high analyzing ability.

In the super-resolution microscope using this double resonanceabsorption process, there have also been proposed a fluorescence labelermolecule which is capable of achieving more effective fluorescenceinhibitation and a suitable timing of light-irradiation.

FIG. 7 illustrates one example of a timing at which a specimen isirradiated with two kinds of lights of wavelengths λ1 and λ2. Asillustrated in FIG. 7, a pulse light shorter than a lifetime of thefirst excited state is employed. The life time of the first excitedstate is a time period for the fluorescence labeler molecule to emitfluorescence. And, the specimen is irradiated at first for a time periodt with the light of the wavelength λ1 and then with the light of thewavelength λ2.

Qualitatively, the irradiation is performed at first for the time periodt with the pulse light of the wavelength λ1 sufficiently shorter thanthe lifetime of the fluorescence labeler molecule in the first excitedstate, thereby to produce a molecule in the first excited state in theobservation area. Immediately after this, an area unnecessary for theobservation is irradiated with the pulse light of the wavelength λ2sufficiently shorter than the lifetime of the first excited state,thereby to excite the molecule in the first excited state to the secondexcited state, thus inhibiting fluorescence.

This process can be further quantitatively explained.

Generally, when the molecule in the ground state is to be excited withthe light of the wavelength λ1 to the first excited state, theexcitation process can be described by the following rate equation.Specifically: the number of molecules per unit area of the molecule dyedto a specimen is designated by N₀; a photon flux of the light of thewavelength λ1 is designated by I_(0;)and the number of molecules in theground state at a time t after irradiation of the light of thewavelength λ1 is designated by N. Moreover, the lifetime of the firstexcited state is designated by τ, and an absorption cross-section upontransition from the ground state to the first excited state by the lightof the wavelength λ1 is designated by a σ₀₁. Then, the rate equation isexpressed in the following form: $\begin{matrix}{\frac{N}{t} = {N_{0}\quad I_{0}\quad \sigma_{01}\quad \frac{\left( {N_{0} - N} \right)}{\tau}}} & {{Equation}\quad 1}\end{matrix}$

If this equation is concretely solved, it is possible to determine thenumber of molecules n in the first excited state per unit volume at thetime t after the light irradiation. That is. $\begin{matrix}{n = {\frac{N_{0}\quad I_{0}\quad \sigma_{01}\quad \tau}{\left( {1 + {I_{0}\quad \sigma_{01}\quad \tau}} \right)} \cdot \left\lbrack {1 - ^{\{{{- {({{I_{0}\quad \sigma_{01}} + \frac{1}{\tau}})}}\quad t}\}}} \right\rbrack}} & {{Equation}\quad 2}\end{matrix}$

wherein n=N₀−N This Eq. 2 can be transformed into Eq. 4 by irradiationwith the light of the wavelength λ1 in such a small quantity as tosatisfy the following Eq. 3:

According to Eq. 3, the value n is substantially proportional to theirradiation time t if the irradiation time of the light of thewavelength λ1 is shorter than the lifetime of the molecule in the firstexcited state and if the photon flux of the light of the wavelength λ1is small.

Next, will be considered the case in which the molecule in the firstexcited state upon irradiation with the light of the wavelength λ2 for atime period T immediately after irradiation of the light of thewavelength λ1 is to be excited to the second excited state.

The photon flux of the light of the wavelength λ2 is designated by I₁;the number of molecules in the first excited state at the time (T+t)after irradiation with the light of the wavelength λ1 is designated byn; and an absorption cross-section upon transition from the firstexcited state to the second excited state by the light of the wavelengthλ2 is designated by σ₁₂. Then, the rate equation on n is expressed inthe following form: $\begin{matrix}{\frac{N}{t} = {{{- \sigma_{12}}\quad I_{1}\quad n} - \frac{n}{\tau}}} & {{Equation}\quad 5}\end{matrix}$

By solving this equation, the value n can be concretely determined as inthe following equation when the irradiation with the light of thewavelength λ1 is made for the time period t and is interrupted and whenthe irradiation with the light of the wavelength λ2 is made immediatelyafter the former irradiation:

n=(I ₀σ₀₁ N ₀ t)·e ^(−(σ) ^(₁₂) ^(I) ^(₁) ^(+1/τ)T)  Equation 6

According to this Eq. 6, on the other hand, the value n is expressed forI₁=0 with no irradiation of the light of the wavelength λ2:

n=(I ₀σ₀₁ N ₀ t)·e ^(T/τ)  Equation 7

As a matter of fact, Eq. 6 indicates the number of molecules in thefirst excited state per unit volume in the area where the fluorescenceis inhibited, and Eq. 7 indicates the number of molecules in the firstexcited state per unit volume in the area where the fluorescence is notinhibited. For a fluorescence yield Φ of the molecule, the fluorescentintensity F1 from the fluorescence inhibited area and the fluorescentintensity F2 from the fluorescence not-inhibited area are given by thefollowing Equations 8 and 9, respectively:

F ₁=Φ(I ₀σ₀₁ N ₀ t)·e ^(−(σ) ^(₁₂) ^(I) ^(₁) ^(+1/τ)T)  Equation 8

F ₂=Φ(I ₀σ₀₁ N ₀ t)·e ^(Tτ/)  Equation 9

A fluorescence inhibition ratio (=F1/F2) is determined to Eq. 10 fromEqs. 8 and 9:

F ₁ /F ₂ =e ^(−τ) ^(₁₂) ^(I) ^(₁) ^(T)  Equation 10

Consequently, if the irradiation with the two kinds of lights of λ1 andλ2 is made at the timings illustrated in FIG. 7, it is possible toinhibit, at the ratio of Eq. 10, the fluorescence from the area notneeded to be observed. According to Eq. 10, the fluorescence can beinhibited at an arbitrary ratio by adjusting the values I₁ and T underthe condition of T<τ.

FIG. 8 illustrates the timing for measuring a fluorescence intensity tobe emitted from the observation area. Basically, the measuring timing ofthe fluorescent intensity is that the fluorescence intensity emittedfrom the observation area is measured for an ample time after the end ofthe irradiation with the light of the wavelength λ2. At this measuringtiming, the fluorescence from the observation area can be measured at anexcellent S/N ratio with little fluorescence from the inhibited area.

FIGS. 9 and 10 exemplify the irradiation timing of the specimen with thetwo kinds of lights of the wavelengths λ1 and λ2 and the measuringtiming of the fluorescent intensity from the observation area,respectively. Also with these timings illustrated in FIGS. 9 and 10, itis possible to realize the super-resolution microscope effectively.

In any of these timings of FIGS. 8 to 10, however, the time periods tand T have to be shorter than the time period τ (t, T<τ). This isbecause if t, T>τ on the contrary, the molecule in the first excitedstate is deexcited to the ground state during the irradiations with thetwo kinds of lights λ1 and λ2, thereby making the fluorescence itselffrom the observation area disappear. For t, T≧τ, the irradiations of thetwo kinds of lights λ1 and λ2 could be made simultaneously, asillustrated in FIG. 11, and the intensity of the fluorescence emittedfrom the observation area could be simultaneously measured. In thiscase, however, the two kinds of excited intense lights λ1 and λ2 mightmove into the detector during the fluorescence measurement.

It is, therefore, desired that the specimen is irradiated under thecondition of t, T<τ with the two kinds of lights λ1 and λ2 at thetimings illustrated in FIGS. 8 to 10. Although the S/N ratio is more orless degraded, the irradiations of the light λ1 and λ2 may also beeffected at absolutely the same timing.

When the specimen is irradiated with the two kinds of lights λ1 and λ2under the aforementioned conditions and at the aforementioned timings,it is necessary to measure the fluorescence emitted from the observationarea immediately after the end of the irradiation by means of adetector. At that time, there are required to prepare gate signals by acommercially available general-purpose logic circuit and fetch theoutput electric signals of the detector in the memory of a personalcomputer.

Basically, as illustrated in the time charts of FIGS. 7 to 10, it raisesthe effect with fact that the fluorescent lifetime of the molecules tobe dyed is longer than the pulse width. In the commercially availablegeneral-purpose logic circuit, however, since the switching rate isabout 1 nsec, the time period τ itself is desired to exceed 1 nsecs. Inother words, unless the fluorescent lifetime exceeds 1 nsec, thefluorescent phenomenon from the observation area ends before thedetector and the measurement circuit become active, so that themeasurements cannot be made. Thus, the fluorescence labeler molecule todye the specimen is required to have a fluorescent lifetime exceeding 1nsec.

On the other hand, noting the effective fluorescent area from which thesignals are to be extracted, it is surely desirable that the fluorescentintensity is weaker in the fluorescence inhibition area, but it isdesired from the view point of the improvement in the S/N ratio that theemission intensity of the effective fluorescence area is stronger. Inshort, the fluorescent intensity is measured from the time when thenumber of molecules in the first excited state just after the excitationwith the light λ1 is sufficient. According to the foregoing Eq. 9, thenumber of excited molecules is attenuated in the manner of anexponential function by the time constant which is determined by theexcitation lifetime. Here, according to the characteristics of theexponential function, if the pulse widths t and T of the light aresufficiently shorter than the lifetime τ of the molecule in the firstexcited state, it is possible to measure fluorescence of a sufficientlystrong intensity, i.e., an effective signal intensity from the moleculein the first excited state just after the excitation with the light λ1.Especially if the time periods t and T are about one tenth of thelifetime of the molecule in the first excited state, the number ofmolecules in the first excited state is as many as 90% of the moleculenumber just after the excitation with the light λ1, so that a sufficientsignal intensity from the effective fluorescent area is achieved.

The optical microscope of the prior art thus far described hasoutstanding usefulness and technical priority in its super-resolutionand analytical ability.

However, the optical microscope of the prior art needs the light of thewavelength λ2 having an intensity sufficient for inhibiting thefluorescence from the first excited state by exciting a molecule fromthe first excited state to the second excited state (hereinafter, thislight will be called the “erase light” and the light of the wavelengthλ1 to excite a molecule from the ground state to the first excited statewill be called the “pump light”). Although the erase light has aslightly lower intensity than a high-intensity laser beam of severalTW/cm2 in the laser scanning type fluorescence microscope using anunresonance two-photon absorption process, it still has a considerablystrong intensity, so that it has raised a problem of influences on thebio-specimen.

This high-intensity laser is excessively strong against the biologicalcells of a specimen. Especially in case where a measurement for a longtime is required, influence by heat reserve or absorption of multiplephotons in the sample is very serious. Thus, such influence has to beminimized.

Moreover, wavelengths of the pump light and the erase light have to falloutside of the absorption band of the biological cells.

Furthermore, in order to realize a resolution as theoreticallyestimated, a beam of the erase light condensed on a specimen surface isrequired to have a zero intensity distribution where an intensity at itscentral portion is zero and to have an axially symmetric shape(hereinafter , this beam will be called the “hollow beam”). This isbecause disturbance in the intensity distribution leads as it is todeterioration in the resolution of the microscope.

A laser is frequently used as a light source for the erase light, and inorder to achieve a theoretical beam as mentioned above, it is a majorpremise that the laser must have a satisfactory beam profile, meaningthat the beam having an intensity distribution symmetric with respect toan optical axis is desirable.

For example, a dye laser, as used as a light source in the prior art,has a beam shape which is close to triangle and an intensitydistribution which is not uniform. As a result, the beam shape condensedon the specimen surface is not the expected hollow beam, but a deformedbeam pattern, thereby causing deterioration in the resolution orreduction in the image quality of a microscope image. In addition, therehas been proposed that the reduced image by a minute zonal aperture isused as the hollow beam. If this zonal aperture is utilized, however, itis difficult to make an optical alignment or to adjust the focal point.It thus takes a seriously long time to obtain a satisfactory image andneeds a skillful technique.

Accordingly, in order to achieve the function of the super-resolutionmicroscope sufficiently, an optical technique for solving those problemshas been required.

Further, from the practical aspect, an excellent operability is also animportant factor.

The microscope technique of the prior art can be applied to a number offluorescence labeler molecules by synchronizing the light of the lightsource with the resonance wavelength of the pump light or the eraselight of the fluorescence labeler molecule by means of a dye laser or anoptical parametric oscillator (OPO).

However, in the dye laser there are problems such as a reduction in thequantity of light due to a deterioration in the dye and a frequent,troublesome dye exchange. The OPO is convenient but remarkablyexpensive. Moreover, the OPO is an extremely precise optical systemrequiring strict managements of humidity and temperature, and anonlinear optical crystal used has a short lifetime and a high price,thereby making it a light source requiring a serious burden ofmaintenance and management on the user.

It is, therefore, preferable that a light source to be used has a fixedwavelength, a simple construction and a reasonable price.

In recent years, there has been developed a micro manipulation techniquewhich can capture and move minute particles under observation of themicroscope by using a laser beam. It has been earnestly desired torealize the microscope system which is enabled to have high operabilityand function by adding the function of the micro manipulation techniqueto the super-resolution microscope.

According to this micro manipulation technique, polarization is producedby condensing a high-intensity laser light to a dielectric particle suchas a polyethylene particle, and then the particle can be captured andmoved by attracting the particle to an area having the strongestelectric field. In this technique, it is preferable to irradiate aparticle with a laser beam in directions as various as possible in orderto stably capture a specific particle.

However, for these laser beam irradiations in the various directions,many laser light sources and complex mirror optical systems arerequired, thereby making the move operation extremely difficult althoughthe capture in one space is possible.

On the other hand, by the irradiation with the laser condensed beam of100 MW/cm² or more in one direction, the specific particle can becaptured in one space and can be spatially moved with scanning of thebeam. Nevertheless, the specimen is continuously exposed to the laserbeam of a high intensity so that it is seriously damaged. This raisesproblems that biological cells are photosensitively killed and that achemical change occurs due to a dissociation or photo-chemical reactionof the molecule themselves.

The invention of this application has been provided in view of thebackground thus far described and has an object to provide a novelmicroscope system which has a capability to condense an erase light forexciting a molecule in the first excited state to the second excitedstate with an excellent beam profile by using a simple, compact opticalsystem and also has a high stability and operability and an excellentsuper-resolution. Also provided is a novel microscope system which has amicro manipulator function to capture and move specimen particles byusing the erase light of a hollow beam without damaging the specimen.

OBJECTS OF THE INVENTION

In order to solve the above-especified problems, the invention of thisapplication provides a microscope system.

According to an aspect of the present invention, a microscope system isprovided which comprises an adjusted specimen and a microscope body,wherein the adjusted specimen is dyed with a molecule which has threeelectron states including at least a ground state and in which anexcited wavelength band from the first electron excited state to thesecond electron excited state overlaps a fluorescent wavelength bandupon deexcitation through a fluorescence process from the first electronexcited state to a vibrational level in the ground state, wherein themicroscope body includes: a light source for a light of a wavelength λ1for exciting the molecule from the ground state to the first electronexcited state; a light source for a light of a wavelength λ2 forexciting the molecule in the first electron excited state to the secondor higher electron excited state; a condensing optical system forcondensing the light of the wavelength λ1 and the light of thewavelength λ2 on the adjusted specimen; overlap means for partiallyoverlapping the irradiation region of the light of the wavelength λ1 andthe irradiation region of the light of the wavelength λ2 on the adjustedspecimen; and an emission detector for detecting an emission upondeexcitation of the excited molecule to the ground state, and wherein aregion of the emission upon deexcitation of the molecule from the firstelectron excited state to the ground state is inhibited by irradiatingthe light of the wavelength λ1 and the light of the wavelength λ2through the overlap means.

According to another aspect of the present invention, a microscopesystem is provided which comprises an adjusted specimen and a microscopebody, wherein the adjusted specimen is dyed with a molecule which hasthree electronic states including at least a ground state, wherein themicroscope body includes: a light source for a light of a wavelength λ1for exciting the molecule from the ground state to the first electronexcited state; a light source for a light of a wavelength λ2 forexciting the molecule in the first electron excited state to the secondor higher electron excited state; a condensing optical system forcondensing the light of the wavelength λ1 and the light of thewavelength λ2 on the adjusted specimen; overlap means for partiallyoverlapping the irradiation region of the light of the wavelength λ1 andthe irradiation region of the light of the wavelength λ2 on the adjustedspecimen; and an emission detector for detecting an emission upondeexcitation of the excited molecule to the ground state, wherein aregion of the emission upon deexcitation of the molecule from the firstelectron excited state to the ground state is inhibited by irradiatingthe light: of the wavelength λ1 and the light of the wavelength λ2through the overlap means, and wherein a beam obtained by condensing thelight of the wavelength λ2 has a phase distribution in which the phaseis shifted by π at a symmetric position with respect to an optical axisof the beam in a plane normal to the optical axis.

According to yet another aspect of the present invention, a microscopesystem is provided which comprises an adjusted specimen and a microscopebody, wherein the adjusted specimen is dyed with a molecule which hasthree electron states including at least a ground state and in which anexcited wavelength band from the first electron excited state to thesecond electron excited state overlaps a fluorescent wavelength bandupon deexcitation through a fluorescence process from the first electronexcited state to a vibrational level in the ground state, wherein themicroscope body includes: a light source for a light of a wavelength λ1for exciting the molecule from the ground state to the first electronexcited state; a light source for a light of a wavelength λ2 forexciting the molecule in the first electron excited state to the secondor higher electron excited state; a condensing optical system forcondensing the light of the wavelength λ1 and the light of thewavelength λ2 on the adjusted specimen; overlap means for partiallyoverlapping the irradiation region of the light of the wavelength λ1 andthe irradiation region of the light of the wavelength λ2 on the adjustedspecimen; and an emission detector for detecting an emission upondeexcitation of the excited molecule to the ground state, wherein aregion of the emission upon deexcitation of the molecule from the firstelectron excited state to the ground state is inhibited by irradiatingthe light of the wavelength λ1 and the light of the wavelength λ2through the overlap means, and wherein a beam obtained by condensing thelight of the wavelength λ2 has a phase distribution in which the phaseis shifted by π at a symmetric position with respect to an optical axisof the beam in a plane normal to the optical axis.

In the aforementioned microscope systems, according to the invention ofthis application:

the excitation wavelength band from the first electron excited state tothe second electron excited state and the excitation wavelength bandfrom the ground state to the first electron excited state are different;

the molecule is a molecule containing one or more of a six-memberedring;

the six-membered ring is a benzene ring or a purine base;

the molecule is a molecule containing one or more of a six-membered ringderivative;

the six-membered ring derivative is a benzene derivative or a purinederivative;

the molecule is any of a xanthene group molecule, a rhodamine groupmolecule, a oxazine group molecule, a cyanine group molecule, a coumaringroup molecule, a oxazole group molecule, a oxadiazole group moleculeand a stilbene group molecule;

the molecule is any of the following molecules:2,2″-dimethyl-p-terphenyl; p-terphenyl (PTP); 3,3′,2″,3′″-tetramethyl-p-quaterphenyl; 2,2′″-demethyl-p-quaterphenyl;2-methyl-5-t-butyl-p-quaterphenyl;2-(4-biphenylyl)-5-(4-t-butylphenyl)-1,3,4-oxiazole (BPBD-365);2-(4-biphenylyl)-phenyl- 1,3,4-oxadiazole;2,5,2″″,5″″-tetrametyl-p-quinquephenyl3,5,3″″,5″″-tetra-t-butyl-p-quinquephenyl;2,5-diphenyloxazole; 2.5-diphenylfuran; PQP (p-quanterphenyl);2,5-bis-(4-biphenylyl)-1,3,4-oxadiazole; p-quaterphenyl-4,4′″-disulfonicacid disodium salt; p-quaterphenyl-4,4′″-disulfonic acid dipotassiumsalt; 4,4′″-bis-(2-butyloctyloxy)-p-quaterphenyl;3,5,3″″,5″″-tetra-butyl-p-sexiphenyl; 2-(1-naphthyl)-5-phenyloxazole;2-(4-biphenylyl)-6-phenylbenzoxazotetrasulfonic acid potassium salt;2-(4-biphenylyl)-6-phenylbenzoxazole-1,3; 4,4′-diphenylstilbene;[1,1′-biphenyl]-4-sulfonic acid, 4,4″,-1,2-ethene-diylbis-,dipotassiumsalt; 2,5-bis-(4-biphenylyl)-oxazole;2,2′-([1,1′-biphenyl]-4,4′-diyldi-2,1-ethenediyl)-bis-benzenesulfonicacid disodium salt; 7-amino-4-methylcarbostyryl;1,4-di[2-(5-phenyloxazole)]benzene; 7-hydroxy-4-methylcoumarin;p-bis(o-methylstylryl)-benzene; benzofuran,2,2′-[1,1′-biphenyl]-4,4′-diyl-bis-tetrasulfonic-acid;7-dimethylamino-4-methylquinolom-2; 7-amino-4-methylcoumarin;2-(p-dimethylaminostyryl)-pyridylmethyl iodide; 7-diethylamonocoumarin;7-diethylamino-4-methylcoumarin;2,3,5,6-1H,4H-tetrahydro-8-methylquinolizino-[9,9a,1-gh]-coumarin;7-diethylamino-4-trifluoromethylcoumarin;7-dimethylamino-4trifluoromethylcoumarin;7-amino-4-trifluoromethylcoumarin;2,3,5,6-1H,4H-tetrahydroquinolizino-[9,9a,1-gh]-coumarin;7-ethylamino-6-methyl-4-trifluoromethylcoumarin;7-ethylamino-4-trifluoromethylcoumarin;2,3,5,6-1H,4H-tetrahydro-9-carboethoxyquinolizino-[9,9a,1-gh]coumarin;2,3,5,6-1H,4H-tetrahydro-9-(3-pyridyl)-quinolizino-[9,9a,1-gh]coumarin;3-(2′-N-methylbenzimidazolyl)-7-N,N-diethylaminocoumarin;2,3,5,6-1H,4H-tetrahydro-9-acetylquinolizino-[9,9a,1-gh]coumarin;N-methyl-4-frifluoromethylpiperidino-[3,2-g]-coumarin;2-(p-dimethylaminostyryl)-benzothiazolylethyliodide;3-(2′-benzimidazolyl)-7-N,N-diethylaminocoumarin; brillantsulfaflavin;3-(2′-benzothiazolyl)-7diethyllaminocoumarin;2,3,5,6-1H,4H-tetrahydro-8trifluoromethylquinolizino-[9,9a,1-gh]coumarin;3,3′-diethyloxacarbocyanine iodide;3,3′-dimethyl-9-ethylthiacarbocyanine iodide; disodium fluorescein(Uranin);9-(o-carboxyphenyl)-2,7-dichloro-6-hydroxy-3H-xanthen-3-on2,7-dichlorofluorescien(Fluorescein 548); Fluorol 555 (Fluorol 7GA);o-(6-amino-3-imino-3H-xanthen-9-yl)-benzonic acid (Rhodamine 560);benzoic acid,2-[ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl],perchlorate(Rhodamine 575); benzoic acid,2-[ethylamino)-3-(ethylimino)-2,7-dimethyl-3X-xanthen-9-yl],ethyl ester,monohydrochloride(Rhodamine 590);1,3′-diethyl-4,2′-quinolyloxacarbocyanine iodide;1,1′-diethyl-2,2′-carbocyanine iodide;2-[6-(diethylamino)-3-(ethylamino)-3H-xanthen-9-yl] benzonic acid(Rhodamine 610);ethanaminium,N-[(6-diethylamino)-9-(2,4-disulfophenyl)-3H-xanthen-3ylidene]-N-ethylhydroxide,inner salt, sodium salt; Malachit Green; 3,3′-diethylthiacarbocyanineiodide; 1,3′-diethyl-4,2′-quinolyloxacarbocyanine iodide;8-(2-carboxyphenyl)2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a,1-bc:9′,9a,1-hi]xantyliumperchlorate (Rhodamine 640);4-dicyanmethylene-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran;3,3′diethyloxadicarbocyanine iodide;8-(2,4-disulfophenyl)-2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a,1-bc:9′, 1-hi]xanthene (Sulforhodamine 640);5,9-diaminobenzo[a]phenoxazonium percrorate;9-diethylamino-5H-benzo[a]phenoxazine-5-one;5-amino-9diethylimino[a]phenoxanium perchlorate;3-ethylamino-7-ethylimino-2,8-dimethylphenoxazine-5-ium perchlorate;8-(trifluoromethyl)-2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a,1-bc:9′,9a,1-hi]perchlorate;1-ethyl-2-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-pyridiniumpercholorate; Carbazine 122;9-ethylamino-5-ethylimino-10-methyl-5H-benzo(a)phenoxazoniumperchlorate; 3-diethylamino-7-diethyliminophenoxazonium perchlorate;3-diethylthiatricarbocyanine iodide; Oxazine 750;1-ethyl-4-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-pyridiniumperchlorate; 1,1′,3,3,3′,3′-hexamethylindodicarcyanine iodide;1,1′-diethyl-4,4′-carbocyanine iodide;2-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-1,3,3-trimethyl-3H-indoliumperchlorate;2-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-3-ethylbenzothoazoliumperchlorate; 1,1′-diethyl-2,2′-dicarbocyanine iodide;1-ethyl-4-(4-(9-(2,3,6,7-tetrahydro1H,5H-benzo(ij)-chinolinozinium))-1,3-butadienyl)-pyridiniumperchlorate; 3,3′-dimethyloxatricarbocyanine iodide;1-ethyl-4-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-quinoliniumperchlorate;8-cyano2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a1-bc:9a′,1-hi]xanthylium perchlorate (Rhodamine 800);2-(6-(4direthylaminophenyl)-2,4-neopentylene-1,3,5)-3-methylbenzothiazoliumperchlorate; 1,1′,3,3,3′,3′-hexamethylindotricarbocyanine iodide; IR125;3,3′-diethylthiatricarbocyanine iodide; IR144;2-(6-(9-(2,3,6,7-tetrahydro-1H,5H-benzo(i,j)-chinolinozinium))-2,4-neopentylene-1,3,5-hexatrienyl)-3-methylbenzothiazoliumperchlorate; 3,3′-diethyl-9,11-neopentylenethiatricarbocyanine iodide;1,1′,3,3,3′-hexamethyl-4,4′,5,5′-dibenzo-2,2′-indotricarbocyanineiodide; 3,3′-diethyl-4,4′,5,5′-dibenzothiatricarbocyamine iodide;1,2′-diethyl-4,4′-dicarbocyanine iodide; IR140;2-(8-(4-p-dimethylaminophenyl)-2,4-neopentylene-1,3,5,7octatetraenyl)-3-methylbenzothiazoliumperchlorate; IR 132;2-(8-(9-(2,3,6,7-tetrahydro-1H,5H,benzo(ij)chinolinozinium))-2,4-neopentylene-1,3,5,7-octatetraenyl)-3-methylbenzothiazoliumperchlorate; IR26; and IR5;

an optical axis of a beam obtained by condensing the light of thewavelength λ1 and the optical axis of the beam obtained by condensingthe light of the wavelength λ2 are coaxial;

the beam obtained by condensing the light of the wavelength λ2 has aphase distribution in which the phase changes continuously from 0 to 2πwhen turned once around the optical axis in a plane normal to theoptical axis;

the beam obtained by condensing the light of the wavelength λ2 has aphase distribution in which the phase changes discontinuously from 0 to2π when turned once around the optical axis in a plane normal to theoptical axis;

the beam obtained by condensing the light of the wavelength λ2 is aBessel beam;

the Bessel beam is a 1-st-order-Bessel-beam;

the beam obtained by condensing the light of the wavelength λ2 is alaser beam having a vibrational mode of any of the Gauss's type,Laguerre's type and Hermitian's type;

any of a gas laser, a solid laser and a semiconductor laser is providedas the light source for the light of the wavelength λ1;

an oscillation wavelength of any of the gas laser, the solid laser andthe semiconductor laser is the wavelength λ1;

a harmonic-wave of an oscillation wavelength of any of the gas laser,the solid laser and the semiconductor laser has the wavelength λ1;

a sum frequency of or a difference frequency between an oscillationwavelength of any of the gas laser, the solid laser and thesemiconductor laser and a harmonic-wave of the oscillation wavelengthhas the wavelength λ1;

any of a gas laser, a solid laser and a semiconductor laser is providedas the light source for the light of the wavelength λ2;

an oscillation wavelength of any of the gas laser, the solid laser andthe semiconductor laser is the wavelength λ2;

a harmonic-wave of an oscillation wavelength of any of the gas laser,the solid laser and the semiconductor laser has the wavelength λ2;

a sum frequency of or a difference frequency between an oscillationwavelength of any of the gas laser, the solid laser and thesemiconductor laser and a harmonic-wave of the oscillation wavelengthhas the wavelength λ2;

the gas laser is any of an excimer laser, a copper vapor laser, an argonlaser, a He—Ne laser, a CO₂ laser, a He—Cd laser and a-nitrogen laser;

the gas laser is of a mode-locked type;

the solid laser is any of a Nd:YAG laser, a Ti sapphire laser, a YLFlaser and a ruby laser;

the solid laser is of a semiconductor-laser-excited type;

the solid laser is of a mode-locked type;

the microscope body has one or more of nonlinear media or wavelengthmodulating element for converting the wavelength of a laser beam fromthe gas laser, the solid laser or the semiconductor laser;

the nonlinear media or the wavelength modulating element is a nonlinearcrystal;

the nonlinear media or the wavelength modulating element is a Ramanshifter;

the light of the wavelength λ1 is prepared by modulating a wavelength ofa fundamental-wave of the gas laser or the solid laser with thenonlinear media or the wavelength modulating element;

the light of the wavelength λ1 is prepared by modulating a wavelength ofa harmonic-wave of the gas laser or the solid laser with the nonlinearmedia or the wavelength modulating element;

the light of the wavelengthλ2 is prepared by modulating a wavelength ofa fundamental-wave of the gas laser or the solid laser with thenonlinear media or the wavelength modulating element;

the light of the wavelength λ2 is prepared by modulating a wavelength ofa harmonic-wave of the gas laser or the solid laser with the nonlinearmedia or the wavelength modulating element;

the condensing optical system for the light of the wavelength λ2 has aphase plate having a refractive-index distribution or anoptical-path-difference distribution which gives, to a beam obtained bycondensing the light of the wavelength of the λ2, a phase differencedistribution in a plane normal to an optical axis of the beam;

the condensing optical system for the light of the wavelength λ2 has azonal optical system;

the condensing optical system for the light of the wavelength λ2 has adiffractive optical system;

the condensing optical system for the light of the wavelength λ2 has anaxicon;

in a resonator of the gas laser, the solid laser or the semiconductorlaser, there is provided at least one of a ring-shaped zonal mirror, azonal diffraction grating, a Fresnel zone plate, a zonal aperture, and aphase plate which gives a phase difference in which electric fieldsaxially symmetric in a plane normal to the optical axis are shifted by xfrom each other;

the microscope body has an emission condensing optical system forcondensing an emission from the molecule to the emission detector;

the emission condensing optical system has a sharp cut filter;

the emission condensing optical system has a notch filter;

the emission condensing optical system has a band-pass filter;

the band-pass filter transmits the emission from the molecule while nottransmitting the light of the wavelength λ1 and the light of thewavelength λ2;

the adjusted specimen is sealed by seal means made of a substancetransmitting the light of the wavelength λ1 and the light of thewavelength λ2;

the adjusted specimen is covered with cover means made of a substancetransmitting the light of the wavelength λ1 and the light of thewavelength λ2;

said substance is synthetic quartz SiO2, CaF2, NaF, Na3AlF6, LiF, MgF2,SiO2, LaF3, NdF3, Al203, CeF3, PbF2, MgO, ThO2, SnO2, La2O3 or SiO;

the microscope body has a continuous-wave laser separately of the lightsources for the light of the wavelength λ1 and the light of thewavelength λ2, and wherein a beam obtained by condensing thecontinuous-wave laser on the adjusted specimen has a phase distributionin which the phase is shifted by π at a position symmetric with respectto an optical axis of the beam in a plane normal to the optical axis;and

the microscope body has means for relatively scanning, on the adjustedspecimen, with a beam obtained by condensing the continuous-wave laseron the adjusted specimen, independently of the beam obtained bycondensing the light of the wavelength λ1 and the beam obtained bycondensing the light of the wavelength λ2.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a conceptional diagram illustrating an electron structure of amolecule.

FIG. 2 is a conceptional diagram illustrating an excitation of themolecule of FIG. 1 to a first excited state by a wavelength λ1.

FIG. 3 is a conceptional diagram illustrating an excitation of themolecule of FIG. 1 to a second excited state by a wavelength λ2.

FIG. 4 is a conceptional diagram illustrating a deexcitation process, asaccompanied by an emission, from the second electron excited state ofFIG. 3 to a ground state.

FIG. 5 is a conceptional diagram illustrating a principle of asuper-resolution microscope for a molecule having a low emission yieldin the second excited state.

FIG. 6 is a diagram illustrating a double resonance absorption process.

FIG. 7 is a diagram illustrating one example of irradiation timings oflights of wavelengths λ1 and λ2 and the number of molecules in the firstexcited state.

FIG. 8 is a diagram illustrating one example of irradiation timings ofthe lights of the wavelengths λ1 and λ2 and measurement timings.

FIG. 9 is a diagram illustrating another example of the irradiationtimings of the lights of the wavelengths λ1 and λ2 and the measurementtimings.

FIG. 10 is a diagram illustrating another example of the irradiationtimings of the lights of the wavelengths λ1 and λ2 and the measurementtimings.

FIG. 11 is a diagram illustrating another example of the irradiationtimings of the lights of the wavelengths λ1 and λ2 and the measurementtimings.

FIG. 12 is a diagram illustrating a deexcitation process of a moleculefrom a high excited state.

FIG. 13 is a diagram illustrating an energy diagram of a molecule whosewavelength range for exciting from a first electron excited state to asecond excited state overlaps a florescent wavelength range upondeexcitation by a florescence process from the first electron excitedstate to a vibrational level of the ground state.

FIG. 14 is a diagram illustrating a molecular structural formula ofRhodamine 6G and relations between individual cross-sections as opticalproperties and wavelengths.

FIG. 15 is a diagram illustrating a coordinate system for expressing aBessel beam.

FIG. 16 is a diagram illustrating a phase distribution of a beam planeof the Bessel beam.

FIG. 17 is a diagram illustrating a two-dimensional intensitydistribution of a 1-st-order-Bessel-beam.

FIG. 18 is a diagram illustrating a phase distribution to be imparted toa condensed beam of an erase light by a phase plate.

FIG. 19 is a diagram illustrating one example of a numerical aperture ofa condensing optical system.

FIG. 20 is a diagram illustrating one example of an erase lightcondensed beam intensity and a fluorescent intensity in the microscopebody of a microscope system of this invention of Example 1.

FIG. 21 is a conceptional diagram illustrating a phase plate having arefractive-index distribution changing discontinuously around an opticalaxis.

FIG. 22 is a conceptional diagram illustrating a phase distribution tobe given to the erase light by a phase plate which has arefractive-index distribution being discontinuous by quartering thedistribution around its optical axis.

FIG. 23 presents, at (a) and (b), a top plan view and a side elevationillustrating a structure and an optical parameter of the phase plate,respectively.

FIG. 24 is a diagram illustrating one example of an erase lightcondensed beam intensity and a fluorescent intensity in the microscopebody of a microscope system of this invention of Example 2.

FIG. 25 is a diagram illustrating one example of an erase lightcondensed beam intensity and a fluorescent intensity in the microscopebody of a microscope system of this invention of Example 3.

FIG. 26 is a construction diagram showing an essential portion of oneexample of the microscope system of this invention.

FIG.27 is a diagram showing one example of a zonal optical system inwhich an annular zonal slit and an ordinary glass lens are combined.

FIG. 28 is a diagram illustrating a reflecting type objective lens asone example of the zonal optical system.

FIG. 29 is a diagram illustrating a Walter type lens as one example ofthe zonal optical system.

FIGS. 30 presents, at (a) and (b), transmission type and reflection typeFresnel zone plates as examples of diffracting optical systems,respectively.

FIG. 31 is a diagram illustrating the transmission diffraction gratinghaving grooves in concentric circles.

FIG. 32 is a diagram illustrating one example of an axicon opticalsystem.

FIG. 33 is a construction diagram showing an essential portion ofanother example of the microscope system of this invention.

FIG. 34 is a construction diagram showing an essential portion ofanother example of the microscope system of this invention.

FIG. 35 is a construction diagram showing an essential portion of oneexample of a laser resonator capable of oscillating a primary Besselbeam directly.

FIG. 36 is a construction diagram showing an essential portion of oneexample of the microscope system of this invention of the case in whicha Nd:YAG laser has the laser resonator shown in FIG. 35.

FIG. 37 presents, at (a), (b), (c), and (d), diagrams illustratingamplitude distributions and intensity distributions on beams of higherorders for n=0, n=1, n=2, and n=3, respectively.

FIG. 38 are diagrams illustrating relations between the wavelengthcharacteristics of various filters and the wavelength characteristics ofdetected lights.

FIG. 39 is a construction diagram showing an essential portion of oneexample of the microscope system of this invention, which is providedwith a filter optical system.

FIG. 40 is a construction diagram showing an essential portion of oneexample of an electric system corresponding to the microscope system ofthis invention of FIG. 34.

Here, reference numerals in the Drawings designate the followingcomponents:

1 Nd:YAG Laser

2 BBO Crystal

3 Half Mirror

4 Raman Shifter

5 Mirror

6 Phase Plate

7 Dichroic Mirror

8 Dichroic Mirror

9 Condensing Objective Lens

10 Two-Dimensional Carriage Stage

100 Adjusted Specimen

11 Fluorescence Condenser Lens

12 Sharp Cut Filter

13 Pin Hole

14 15 Photomultiplier

16 Zonal Slit

17 Glass Lens

18 3-rd harmonics Generator

19 2-nd harmonics Generator

20, 24 Dichroic Mirror

21 Polarizer

22 Condenser Lens

23 Pin Hole

25 Objective Lens

26 Pin Hole

27 Sharp Cut Filter

28 Half Mirror

29 30 Mirror

31 Mercury Lamp

32, 33 Nd:YAG Laser

35, 36 KTP Crystal

37, 38 Half Mirror

39 Raman Shifter

40, 41 Dichroic Mirror

42 Relay Lens

43, 44, 45 Half Mirror

46, 48 Lens

47 Pin Hole

49 Spectrometer

50 CCD Camera Focusing Lens

51 CCD Camera

52 Zonal Mirror

53 Lens

54 Phase Plate

55 Output Mirror

56 Nd:YAG Laser

57 KTP Crystal

58 Notch Filter

59 Band-pass Filter

60 Sharp Cut Filter

61 Pin Hole

62 Shade Box

63 Cover Glass

64 Objective Lens

65 Condenser Lens

66 Personal Computer

67 Frequency Divider

68 Gate & Delay Generator

69 CCD Array

70 Diffraction Grating

71 CCD Camera

72 Frame Memory

73 CRT

74 Video Printer

BEST MODE FOR CARRYING OUT THE INVENTION

In the microscope system of the invention of this application; theadjusted specimen is dye with the molecule which has such threeelectronic states including at least the ground state that theexcitation wavelength band from the first electron excited state to thesecond electron excited state overlaps a fluorescent wavelength bandupon deexcitation by a fluorescent process from the first electronexcited state to a vibrational level of the ground state.

Here will be described in detail this molecule, i.e., the so called“fluorescence labeler molecule” by considering the deexcitation processfrom the high electron excited state.

FIG. 12 conceptionally illustrates a deexcitation process of a molecule.Generally, when the molecule is excited from a ground state S0 to alowermost (=first) excited state S1, the molecules having π electronsare deexcited to S0, emitting a fluorescence at a yield of several tens% at the most. This is fluorescence process. The remainder isdeactivated directly to S0 without any radiation by vibrationalrelaxation, i.e., by internal conversion, but a portion thereof reachesa state T1 of which a spin multiplicity is different and a lifetime isextremely long, that is, performs a inter-system crossing, and thenreturns to S0 from the state T1, emitting a phosphorescence. In theordinary fluorescent microscope, a specimen is dyed with a moleculehaving a high fluorescent yield and this molecule is excited to S1 by anlight irradiation, so that fluorescence from S1 is observed andvisualized.

On the other hand, when the molecule is excited to a second excitedstate S2 higher than S1, it deexcites to the ground state S0 by aremarkably complicated relaxation process, as illustrated in FIG. 12.For example, some of the excited molecules in S2 is deexcited to S0 orT1 by internal conversion or inter-system crossing. The remainingexcited molecules are internally converted to the high vibrational levelin S1, then they reach the lowermost vibrational level in S1. Afterthis, the molecules return to S0 through the aforementioned relaxationprocess from S1.

What should be noted here is that the fluorescent emission yield from anexcited state higher than S2 is extremely low. This is because many ofthe molecules in S2 are either deactivated without any radiationdirectly to S0 or deactivated without any radiation to S0 at aconsiderable ratio after having been internally converted to S1. And,since the molecules having reached the state T1 make no contribution tothe fluorescent process, only a portion of the molecules having reachedS1 emits a fluorescence. This is called the “Krash's law”. Especiallyfor a benzene derivative molecule in a gas phase, little fluorescencefrom S2 is observed.

Generally, the super-resolution microscope using the double resonanceabsorption process makes use of the fact that the fluorescent yield of amolecule from an electron excited state of S2 or higher level than S2 isextremely low, as described hereinbefore.

Here, some molecules have a structure, in which the wavelength band fortransition from S1 to Sn (n=2 or more) overlaps a fluorescent emissionwavelength band upon deexcitation from S1 to the vibrational level S0 bythe fluorescent process, as illustrated in the energy diagram of FIG.13. In the molecule having this electronic structure, there occurs aphenomenon that the fluorescent inhibition effect caused substantiallyby an erase light is intensified.

When exciting the molecule in S0 to S1 by a pump light of a wavelengthλ1 and irradiating an erase light of a resonance wavelength λ2, themolecule in S1 transit to the state Sn, and, at the same time, mostmolecules are deexcited to the higher vibrational level of S0 by inducedemission. At this time, the molecule excited to the state Sn isinhibited from fluorescence. On the other hand, for the molecule of theinduced emission, a light of the same wavelength as that of the eraselight is emitted, so that the intensity of the light of the samewavelength as that of the erase light slightly increases whereas theintensity of the fluorescence of a wavelength other than that of theerase light decreases. It follows that a substantially excellentfluorescent inhibition can be effected so long as the fluorescentemission of a wavelength other than that of the erase light is beingmonitored.

A molecule, of which the wavelength band upon transition from S1 to thestate Sn overlaps the fluorescent emission wavelength band from S1 asdescribed, is employed as a molecule for dyeing a specimen in themicroscope system of the invention of this application. As a result, thefluorescent inhibition effect to which the aforementioned inducedemission make contributions is added, thereby improving further thesuper-resolution of the microscope body. At the same time, thefluorescent inhibition can be easily caused even for a low intensity ofthe erase light, thus damage on the specimen to be observed can bereduced.

As an example of the molecule having the aforementioned opticalcharacteristics, there is a molecule of a Rhodamine group belonging to axanthene group.

FIG. 14 illustrates a molecular structural formula of Rhodamine 6G thatis a molecule of Rhodamine group and a relation between eachcross-section and a wavelength as optical properties (E. Sahar & D.Treves: IEEE J. Quantum Electron., QE-13,962 (1977)). In FIG. 14, σ_(a)indicates an absorption cross-section from S0 to S1; σ_(e) indicates aninduced emission cross-section from S1 to S0; σ_(a′)indicates anabsorption cross-section from S1 to Sn; and σ_(T) indicates anabsorption cross-section from T1 to Tn.

As illustrated in FIG. 14, the resonance wavelength from S0 to S1extends around about 530 nm (as should be referred to σ_(a))., but theresonance wavelength from S1 to Sn extends around about 500 to 600 nm(as should be referred to σa*). And, the fluorescent emission bandextends to the region of about 530 to 650 nm (as should be referred toσ_(e)), overlapping the resonance wavelength region from S1 to Sn.Moreover, it is found that a special wavelength band exists at about 530to 600 nm. Specifically, the light of this wavelength band cannot excitethe molecule from S0 to S1 but can effect the double resonanceabsorption process and the induced emission from S1 to Sn.

Generally, for not only this Rhodamine 6G but also a molecule ofRhodamine group, generally, the wavelength upon transition from S1 to Snoverlaps the fluorescent wavelength band upon deexcitation from S1 tothe vibrational level of S0 through fluorescent process.

Also, for a molecule of coumarin group, the wave length band upontransition from S1 to Sn overlaps the fluorescent emission wavelengthband from S1. For example, Coumarin 500 that is a coumarin groupmolecule, has a resonance wavelength from S0 to S1 extending around 260nm, a resonance wavelength from S1 to Sn extending around 355 nm, and afluorescent emission region extending to the region of 320 to 460 nm.

Basically, like the aforementioned Rhodamine group molecule or coumaringroup molecule, a molecule having such optical properties contains oneor more of a six-membered ring such as a benzene ring and a nitrogenbase (i.e., purine base), or a six-membered ring derivative such as abenzene derivative and a purine derivative, and is exemplified by notonly the Rhodamine group molecule or coumarin group molecule but also axanthene group molecule, oxazine group molecule, cyanine group molecule,oxazole group molecule, oxadiazole group molecule or stilbene groupmolecule.

The following molecules are examples of such molecule:

2,2″-dimethyl-p-terphenyl; p-terphenyl (PTP);3,3′,2″,3′″-tetramethyl-p-quaterphenyl; 2,2′″-demethyl-p-quaterphenyl;2-methyl-5-t-butyl-p-quaterphenyl;2-(4-biphenylyl)-5-(4-t-butylphenyl)-1,3,4-oxiazole (BPBD-365);2-(4-biphenylyl)-phenyl-1,3,4-oxadiazole;2,5,2″″,5″″-tetramethyl-p-quinquephenyl;3,5,3″″,5″″-tetra-t-butyl-p-quinquephenyl; 2,5-diphenyloxazole;2,5-diphenylfuran; PQP (p-quanterphenyl);2,5-bis-(4-biphenylyl)-1,3,4-oxadiazole; p-quaterphenyl-4,4′″-disulfonicacid disodium salt; p-quaterphenyl-4,4′″-disulfonic acid dipotassiumsalt; 4,4′″-bis-(2-butyloctyloxy)-p-quaterphenyl;3,5,3″″,5″″-tetra-butyl-p-sexiphenyl; 2-(1-naphthyl)-5-phenyloxazole;2-(4-biphenylyl)-6-phenylbenzoxazotetrasulfonic acid potassium salt;2-(4-biphenylyl)-6-phenylbenzoxazole-1,3; 4,4′-diphenylstilbene;[1,1′-biphenyl]-4-sulfonic acid, 4,4″,-1,2-ethenediylbis-,dipotassiumsalt; 2,5-bis-(4-biphenylyl)-oxazole;2,2′-([1,1′-biphenyl]-4,4′-diyldi-2,1-ethenediyl)-bis-benzenesulfonicacid disodium salt; 7-amino-4-methylcarbostyryl;1,4-di[2-(5-phenyloxazole)]benzene; 7-hydroxy-4-methylcoumarin;p-bis(o-methylstylryl)-benzene;benzofuran,2,2′-[1,1′-biphenyl]-4,4′-diyl-bis-tetrasulfonic-acid;7-dimethylamino-4-methylquinolom-2; 7-amino-4-methylcoumarin;2-(p-dimethylaminostyryl)-pyridylmethyl iodide; 7-diethylamonocoumarin;7-diethylamino-4-methylcoumarin;2,3,5,6-1H,4H-tetrahydro-8-methylquinolizino-[9,9a,1-gh]-coumarin;7-diethylamino-4-trifluoromethylcoumarin;7-dimethylamino-4-trifluoromethylcoumarin;7-amino-4-trifluoromethylcoumarin;2,3,5,6-1H,4H-tetrahydroquinolizino-[9,9a,1-gh]-coumarin;7-ethylamino-6-methyl-4-trifluoromethylcoumarin;7-ethylamino-4-trifluoromethylcoumarin;2,3,5,6-1H,4H-tetrahydro-9-carboethoxyquinolizino-[9,9a,1-gh]coumarin;2,3,5,6-1H,4H-tetrahydro-9-(3-pyridyl)-quinolizino-[9,9a,1-gh]coumarin;3-(2′-N-methylbenzimidazolyl)-7-N,N-diethylaminocoumarin;2,3,5,6-1H,4H-tetrahydro-9-acetylquinolizino-[9,9a,1-gh]coumarin;N-methyl-4-frifluoromethylpiperidino-[3,2-g]-coumarin;2-(p-dimethylaminostyryl)-benzothiazolylethyl iodide;3-(2′-benzimidazolyl)-7-N,N-diethylaminocoumarin; brillantsulfaflavin;3-(2′-benzothiazolyl)-7-diethytaminocoumarin;2,3,5,6-1H,4H-tetrahydro-8-trifluoromethylquinolizino-[9,9a,1-gh]coumarin;3,3′-diethyloxacarbocyanine iodide;3,3′-dimethyl-9-ethylthiacarbocyanine iodide; disodium fluorescein(Uranin);9-(o-carboxyphenyl)-2,7-dichloro-6-hydroxy-3H-xanthen-3-on2,7-dichlorofluorescien(Fluorescein 548); Fluorol 555 (Fluorol 7GA);o-(6-amino-3-imino-3H-xanthen-9-yl)-benzonic acid (Rhodamine 560);benzoic acid,2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl],perchlorate(Rhodamine 575); benzoic acid,2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3X-xanthen-9-yl],ethylester, monohydrochloride (Rhodamine 590);1,3′-diethyl-4,2″-quinolyloxacarbocyanine iodide;1,1′-diethyl-2,2′-carbocyanine iodide;2-[6-(diethylamino)-3-(ethylamino)-3H-xanthen-9-yl]benzonic acid(Rhodamine 610);ethanaminium,N-[(6-diethylamino)-9-(2,4-disulfophenyl)-3H-xanthen-3-ylidene]-N-ethylhydroxide,inner salt, sodium salt; Malachit Green; 3,3′-diethylthiacarbocyanineiodide; 1,3′-diethyl-4,2′-quinolythiacarbocyanine iodide;8-(2-carboxyphenyl)-2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a,1-bc:9′,9a′,1-hi]xantyliumperchlorate (Rhodamine 640);4-dicyanmethylene-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran;3,3′-diethyloxadicarbocyanine iodide;8-(2,4-disulfophenyl)-2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a,1-bc:9′,1-hi]xanthene(Sulforhodamine640); 5,9-diaminobenzo[a]phenoxazonium percrorate;9-diethylamino-5H-benzo(a)phenoxazine-5-one;5-amino-9-diethylimino[a]phenoxazonium perchlorate;3-ethylamino-7-ethylimino-2,8-dimethylphenoxazine-5-ium perchlorate;8-(trifluoromethyl)-2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a,1-bc:9′,9a,1-hi]xantbyliumperchlorate;1-ethyl-2-(4-(p-dirmethylaminophenyl)-1,3-butadienyl)-pyridiniumperchlorate; Carbazine 122;9-ethylamino-5-ethylimino-10-methyl-5H-benzo(a)phenoxazoniumperchlorate; 3-diethylamino-7-diethyliminophenoxazonium perchlorate;3-diethylthiatricarbocyanine iodide; Oxazine 750;1-ethyl-4-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-pyridiniumperchlorate; 1,1′,3,3,3′,3′-hexamethylindodicarcyanine iodide;1,1′-diethyl-4,4′-carbocyanine iodide;2-(4-(p-dirmethylaminophenyl)-1,3-butadienyl)-1,3,3-trimethyl-3H-indoliumperchlorate;2-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-3-ethylbenzothoazoliumperchlorate; 1,1′-diethyl-2,2′-dicarbocyanine iodide;1-ethyl-4-(4-(9-(2,3,6,7-tetrahydro-1H,5H-benzo(i,j)-chinolinozinium))-1,3-butadienyl)-pyridiniumperchlorate; 3,3′-dimethyloxatricarbocyanine iodide;1-ethyl-4-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-quinoliniumperchlorate;8-cyano-2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9al-bc:9a′,1-hi]xanthyliumperchlorate (Rhodamine 800);2-(6-(4-dimethylaminophenyl)-2,4-neopentylene-1,3,5)-3-methylbenzothiazoliumperchlorate; 1,1′,3,3,3′,3′-hexamethylindotricarbocyanine iodide; IR125;3,3′-diethylthiatricarbocyanine iodide; IR144;2-(6-(9-(2,3,6,7-tetrahydro-1H,5H-benzo(i,j)-chinolinozinium))-2,4-neopentylene-1,3,5-hexatrienyl)-3-methylbenzothiazoliumperchlorate; 3,3′-diethyl-9,11-neopentylenethiatricarbocyanine iodide;1,1′,3,3,3′-hexamethyl-4,4′,5,5′-dibenzo-2,2′-indotricarbocyanineiodide; 3,3′-diethyl-4,4′,5,5′-dibenzothiatricarbocyamine iodide;1,2′-diethyl-4,4′-dicarbocyanine iodide; IR140;2-(8-(4-p-dimethylaminophenyl)-2,4-neopentylene-1,3,5,7-octatetraenyl)-3-methylbenzothiazoliumperchlorate; IR132;2-(8-(9-(2,3,6,7-tetrahydro-1H,5H,benzo(i,j)chinolinozinium))-2,4-neopentylene-1,3,5,7-octatetraenyl)-3-methylbenzothiazoliumperchlorate; IR26; and IR5.

In the above molecules, for example,7-ethylamino-4-trifluoromethylcoumarin (C₁₂H₁₀NO₂F₃: Coumarin 500) thatis a coumarin group molecule has an excitation wavelength band from S0to S1 extending around 266 nm; an excitation wavelength band from S1 toS2 extending around 532 nm, and a fluorescent wavelength band at 532 nm.

These wavelengths 266 nm and 532 nm correspond to the 4-th harmonics andthe 2-nd harmonics of the YAG laser, respectively, so that they can beeasily produced by modulating their fundamental-wave or harmonic-wavewith a nonlinear crystal such as a BBO crystal or KTP crystal as awavelength convertible nonlinear medium or a wavelength modulatingelement.

Accordingly, in the microscope system of this invention, if the coumaringroup molecule is used as the specimen dyeing molecule and the YAG laseris employed as a light source for the pump light and a light source forthe erase light in the microscope body, the microscope system not onlycan achieve an excellent super-resolution but also can have superiorworkability and optical performance to those of the microscope of theprior art using a dye laser.

Specifically, the wavelength to be employed can be determined merely byinitially setting the angle of the nonlinear crystal basically, so thata desired excitation wavelength can be easily emitted without anytroublesome wavelength adjustment. Furthermore, there is neitherfluctuation nor reduction of the laser power due to the degradation ofthe dye and the energy conversion efficiency is excellent, so that thereis no need to use a laser of a high power output thus its light sourcecan be small-sized and inexpensive and also damage on the biologicalspecimen can be reduced.

For the beam profile of the YAG laser, there has been established thetechnique of emitting a satisfactory Gaussian beam having an adjustedphase plane, so that an erase light of an excellent hollow beam typewith optical-axis symmetry can be produced.

As such YAG laser, for example, a commercially available mode-locked YAGlaser having a pulse width of 20 psecs or less and a repetitionfrequency of 100 MHz can be used.

Similarly, no matter whether the dyed molecule might be those of thexanthene group or Rhodamine group, on the other hand, the pump light andthe erase light can be produced by using the YAG laser and the nonlinearcrystal.

For Rhodamine 6G, for example, the resonance wavelength from S0 to S1extends around 530 nm, and the resonance wavelength from S1 to Snextends around 500 to 600 nm, as has been described hereinbefore. Thepump light around 530 nm can be coped with by the 2-nd harmonics of theYAG laser. The erase light around 500 to 600 nm can be easily producedby using the Raman effect (or the so-called “Raman shifter”).of thenonlinear crystal. By filtering the light of 532 nm of the 2-ndharmonics of the YAG laser through the Raman shifter, for example, it ispossible to produce a laser beam around 560 nm, as shifted by about 30nm to a longer wave side, in a conversion efficiency of about 20%.

Further, a laser beam having a sum frequency or a difference frequencybetween fundamental-wave and harmonic -wave, as produced by using thenonlinear crystal, of the YAG lasers can also be employed as the pumplight or the erase light.

It is quite natural that not only the YAG laser but also a gas laser, asolid laser or a semiconductor laser of a fixed wavelength, a variety ofnonlinear media or a wavelength modulation element can be used inconformity to the aforementioned individual molecules.

For example, as the gas laser, an excimer laser, a copper vapor laser,an argon laser, a He—Ne laser, a CO₂ laser, a He—Cd laser or a nitrogenlaser may be used, and as the solid laser, a Nd:YAG laser, a Ti sapphirelaser, a YLF laser or a ruby laser may be used.

Of these individual lasers, the mode-locked type laser has a highrepetition frequency and a pulse amplitude of several tens psecs or lessand is the light source which is more suited for the super-resolutionmicroscope body in the microscope system of this invention.

In recent years, a semiconductor-laser-excited and mode-locked typesold-state laser of small size, high luminance, short pulse and highrepetition is realized at a low cost and in a maintenance-free manner,so that this solid-state laser has all the conditions for exploiting thefunctions of the super-resolution microscope body sufficiently.

Each of the aforementioned gas laser, solid laser and semiconductorlaser is used as the light source for the pump light of the wavelengthλ1 and as the light source for the light of the wavelength λ2 to producethe pump light and the erase light with their vibration wavelength orharmonic-wave, or with the sum frequency or differential frequencybetween the vibration wavelength and the harmonic-wave being set at thewavelength λ1 and the wavelength λ2. On the other hand , the pump lightof the wavelength λ1 and the erase light of the wavelength λ2 can alsobe produced by modulating the wavelength of the fundamental-wave orharmonic-wave of those various light sources with the various nonlinearmedia such as nonlinear crystals or the wavelength modulating elements.

Here, in order that the super-resolution microscope using thefluorescent inhibiting effect may have a theoretical resolution, thebeam to be produced by condensing the erase light which is the light ofthe wavelength λ2 on the specimen surface has to be shaped to have azero optical intensity at a central portion thereby to leave afluorescent region at the central portion in the irradiation region, ashas been described hereinbefore.

In this invention, the beam to be produced by condensing the erase lightis given a phase distribution, in which the phase is shifted by π at anobject position with respect to its optical axis in a plane normal tothe optical axis so that the optical intensity at the central portionmay be zero.

A Bessel beam is suited for the beam having such phase distribution.

If a coordinate system illustrated in FIG. 15 is assumed, for example,the Bessel beam can be expressed in the form of the following Equation:

I(x,y)=|E(x,y)|² |E ₀∫₀ ^(2π)exp[ik sin θ(x cosφ+y sinφ).exp(−imφ)dφ]dφ| ²  Equation 11

In this equation, E (x, y) indicates a field vector, and EO indicates anamplitude of the field vector. If here it is assumed that m=1, the aboveequation expresses a 1-st-order-Bessel-beam. This 1-st-order-Bessel-beamhas a unique point on which the field intensity is zero on the opticalaxis, and is achieved in fact by solving the following wave equation ofelectromagnetic waves: $\begin{matrix}{{{\Delta \quad E\quad \left( {x,y} \right)} - {\mu \quad ɛ\quad \frac{{\partial E}\quad \left( {x,y} \right)}{\partial t}}} = 0} & {{Equation}\quad 12}\end{matrix}$

If a boundary condition axially symmetric with respect to an axis isgiven, Eq. 12can be rewritten with a cylindrical coordinate system (r,φ, z) in the following form: $\begin{matrix}{{{\frac{1}{r}\quad \frac{\partial\quad}{\partial r}\quad \left( \frac{\partial E}{\partial r} \right)} + {\frac{1}{r^{2}}\quad \frac{\partial{\,^{2}E}}{\partial{\,^{2}\varphi}}} + \frac{\partial{\,^{2}E}}{\partial{\,^{2}z}} + {k^{2}\quad E}} = 0} & {{Equation}\quad 13}\end{matrix}$

Here, k indicates a wave number, and a solution of Eq. 11 to beexpressed by a overlap of the Bessel function can be obtained if thecoordinate system illustrated in FIG. 15 is used again.

FIG. 16 illustrates a phase distribution of a beam plane, i.e., a pupilplane of the 1-st-order-Bessel-beam for m=1. As apparent from FIG. 16,the 1-st-order-Bessel-beam has a phase distribution, in a plane normalto its optical axis, changing continuously from 0 to 2 π when turnedonce around the optical axis, and the phases of electric fields axiallysymmetric are shifted by π from each other. As a result, it is foundthat the electric fields completely cancel each other on the opticalaxis to zero so that the Bessel beam has-a singular point at which thefield intensity is zero on the optical axis.

In short, the microscope system of this invention is enabled to leavethe fluorescent region at the central portion of the irradiation regionthereby to have a theoretical resolution by making the condensed beam ofthe erase light into the Bessel beam, especially, the1-st-order-Bessel-beam, for example with the condensing optical system.

Moreover, this Bessel beam is a quasi-nondiffractive beam which will notapparently diffuse, as illustrated in a profile that is atwo-dimensional intensity distribution exemplified in FIG. 17.

The microscope of the prior art employs an objective lens having a largenumerical aperture so as to enhance the resolution by reducing thediffractive limit, thus its focal depth is extremely reduced, making thefocusing operation difficult. In the microscope system of thisinvention, however, the Bessel beam as the condensing beam of the eraselight is a quasi-nondiffractive beam so that its focal depth issubstantially enlarged, lightening the focusing burden. In themicroscope body, moreover, the resolution is dominated by the eraselight so that the operability is enhanced without lowering theresolution.

It is made possible by a condensing optical system using the existingoptical element to easily form the condensed beam of the erase light, asexemplified by the Bessel beam having the aforementioned opticalcharacteristics, that is, the condensed beam, having a boundarycondition axially symmetric with respect to the optical axis and alsohaving a phase distribution in which a phase is shifted by π at theposition symmetric with respect to the optical axis.

In order to give the boundary condition axially symmetric with respectto the optical axis, the condensing optical system is preferablyprovided with a zonal optical system such as a reflecting objective lenshaving a zonal pupil, that is an optical system having a zonal aperture,or a diffractive optical system such as a Fresnel zone plate, or anaxicon.

Additionally, in order to give the phase distribution in which the phaseis shifted by π at a position symmetric with respect to the opticalaxis, the condensing optical system may also be provided with a phaseplate having such a refractive-index distribution oroptical-path-difference distribution as will give a phase differencedistribution in a plane normal to the optical axis.

For example, the condensed beam having the phase distribution changingcontinuously from 0 to 2 π when turned once around the optical axis inthe plane normal to the optical axis, like a complete1-st-order-Bessel-beam, is formed by changing its refractive-indexdistribution or optical-path-difference distribution continuously from 0to 2 π when the phase plate makes one turn in a direction to turn aroundthe optical axis in the plane normal to the optical axis of the beam, asillustrated in FIG. 18.

On the other hand, the condensed beam which is not the complete1-st-order-Bessel-beam but the condensed beam can be formed, if it hasthe phase distribution changing discontinuously from 0 to 2 π whenturned once around the optical axis in the plane normal to the opticalaxis, even if the refractive-index distribution oroptical-path-difference distribution changes discontinuously from 0 to 2π. The light source for the erase light may be given a function to formsuch condensed beam.

Further, the aforementioned boundary condition can be given to convertthe erase light itself directly into the 1-st-order-Bessel-beam byinserting, into a resonater of a laser as the erase light source, atransmission type zonal diffraction grating, a ring-shaped zonal mirror,a Fresnel zone plate, a zonal aperture, or a phase plate giving a phasedifference in which electric fields axially symmetric with respect tothe electric field in a plane normal to the optical axis are shifted byπ from each other.

Furthermore, by adjusting the boundary condition of a resonator of alaser as the erase light source thereby to form an axially symmetricmode pattern, such as TEM11, having a zero intensity on the optical axisand then by condensing this mode pattern with the diffracting opticalsystem having the aforementioned zonal aperture, the laser beam of anyvibrational mode such as Gauss's type, Laguerre's type or Hermitian'stype having a higher order mode pattern, for example, may be convertedinto the condensed beam of the erase light.

On the other hand, the microscope body of the microscope system of thisinvention can also have a micro-manipulator function capable to captureand move a specimen particle with a hollow beam.

When the hollow beam is irradiated, the specimen particle is absorbedinto the region of a high laser intensity, and their stable pointbecomes to the hollow portion of the condensed point position of thehollow beam, so that the particle is completely sealed and captured inthe hollow beam. In the microscope body of this invention, the specimento be captured is hardly irradiated with the laser beam so that itsdamage can be inhibited. Moreover, the spatial movement can be realizedby scanning the beam by the optical system such as the galvano-mirror asin the ordinary laser beam.

In addition, the laser intensity proper for the capture is known to beabout several tens MW/cm²; and this intensity is substantially equal tothe maximum intensity of the erase light used in the microscope body ofthe microscope system of this invention, so that the aforementionederase light source and condensing optical system can be used as theyare.

Accordingly, in the microscope system of this invention, the specimencan be captured and moved without any light irradiation damage by usingthe hollow beam, so that a high-grade micro-manipulator function can beachieved.

Embodiments of this invention will be described with its embodimentswith reference to examples along with the accompanying drawings.

EXAMPLES Example 1

In the microscope system of this invention, the adjusted specimen isdyed with the Rhodamine 6G, and the microscope body is provided with theYAG laser as the light source for the pump light (i.e., the light of thewavelength λ1 for exciting the Rhodamine 6G from the ground state S0 tothe first excited state S1).and the light source for the erase light(i.e., the light of the wavelength λ2 for exciting the Rhodamine 6G inthe first excited state S1 to the second excited state S2). Themicroscope body is further provided with the Raman shifter as thewavelength modulating element of the YAG laser.

The Rhodamine 6G can realize, as described hereinbefore, an excellentfluorescent inhibition by double resonance absorption and inducedemission, as described hereinbefore, and its excitation wavelength λ1from S0 to S1 is 532 nm and its excitation wavelength λ2 from S1 to S2is 560 nm. Hence, the 2-nd harmonics light (=532 nm) of the YAG laser isemployed as the pump light and the light made by modulating the 2-ndharmonics to 560 nm with the Raman shifter is employed as the eraselight.

The following Table 1 shows the optical parameters of the Rhodamine 6Gat 532 nm and 560 nm:

TABLE 1 Absorption Cross-Section of σ₀₁: 4 × 10⁻¹⁶ cm² S0 → S1 (532 nm)Absorption Cross-Section of σ₁₂: 1 × 10⁻¹⁶ cm² S1 → S2 (560 nm)Fluorescent Emission Area σ f: 2 × 10⁻¹⁶ cm² (560 nm) FluorescentLifetime τ: 3 nsecs or more

Moreover, the beam to be produced by condensing the erase light with theuse of the condensing optical system of the microscope body has a phasedistribution in which the phase is shifted by π at a position symmetricwith respect to the optical axis in a plane normal to the optical axisand in which the phase changes continuously from 0 to 2 π when turnedonced around the optical axis. And, the condensing optical system isprovided with a phase plate having the refractive-index distribution oroptical-path-difference distribution, as illustrated in FIG. 18, capableof giving such phase distribution to the condensed beam of the eraselight.

The level of the resolution of the microscope body in such microscopesystem of this invention can be expressed by Eq. 11 that is the generalEquation of the 1-st-order-Bessel-beam mentioned hereinbefore.

This resolution level can be-rewritten in the form of convolutioncalculations, as expressed by the following Equation, if the numericalaperture of the condensing optical system of the erase light isspecifically given as illustrated in FIG. 19:

I(x,y)=|E(x,y)|² |E ₀∫∫_(X′) _(²) _(+Y) _(²) _(≦a) _(²) PSF(x−x′,y−y′)exp[−iφ(x′,y′)]x′dy′| ²  Equation 14

In this Equation, PSF(x, y) indicates a two-dimensional point-imagedistribution function of the optical system, φ (x, y)indicates the phasedistribution; and a indicates the radius of integration area.

When the condensing optical system is a zonal optical system, PSF(x, y)is given by the following Equation: $\begin{matrix}{{{PSF}\quad \left( {x,y} \right)} = {\frac{2J_{1}\quad \left( {2\quad \pi \quad \xi} \right)}{2\quad \pi \quad \xi} - \frac{2\quad \rho_{0}\quad J_{1}\quad \left( {2\quad \pi \quad \xi} \right)}{2\quad \pi \quad \xi}}} & {{Equation}\quad 15}\end{matrix}$

Here, ρ₀ indicates a shading factor of the pupil of the condensingoptical system, and ξ is expressed by the following Equation:$\begin{matrix}{\xi = {\frac{NA}{\lambda}\sqrt{x^{2} + y^{2}}}} & {{Equation}\quad 16}\end{matrix}$

Here, NA indicates the numerical aperture of the condensing opticalsystem, and x indicates the wavelength of the erase light.

In Eq. 15, ρ₀ takes a value between 0 to 1 and, at 0, indicates PSF(x,y) of the condensing optical system not being the zonal optical system.

Here, if I (x, y) given by Eq. 14 is substituted into Eq. 8, thefluorescent intensity F1(x, y) when the specimen surface havinghomogeneously distributed molecule in S1 is irradiated with the eraselight can be determined by the following Equation:

F ₁(x,y)=Φ(I ₀σ₀₁ N ₀ t)·e ^(−(σ) ^(₁₂) ^(I(x,y)+1/τ)T)  Equation 17

When the induced emission contributes to the fluorescent inhibition,moreover, Eq. 17 turns into Eq. 18 as the induced emission cross-sectionbeing σ_(f):

F ₁(x,y)=Φ(I ₀σ₀₁ N ₀ t)·e ^(−((σ) ^(₁₂) ^(+σ) ^(_(ƒ)+I(x,y)+1/τ)T))  Equation 1

Consequently, when the fluorescence labeler molecule is the Rhodamine 6Gand the condensed beam of the erase light has the aforementioned phasedistribution, it is possible to estimate, by using this Eg.18, the levelof spatial resolution of detecting the fluorescence to be emitted upondeexcitation of the Rhodamine 6G the first excited state to the groundstate Hence, the spatial resolution to be achieved in detection by themicroscope body of the microscope system of this invention in thepresent Example 1 was determined from Eq. 18.

Table 2 shows the environmental parameters used for this determination:

TABLE 2 Numerical Aperture of Condenser 0.75 Lens Shading Factor ofOptical 0.95 System Pupil Pulse Width of Laser Beam 150 psecs Wavelengthof Pump Light 532 nm Wavelength of Erase Light 560 nm Photon Flux ofErase Light 9.6 × 10²⁵ photons/sec/cm² Laser Intensity of Erase Light 34MW/cm²

The optical parameters of the Rhodamine 6G adopted the values of theaforementioned Table 1.

Here, the photon flux of the pump light is not designated, but thediscussions on the yield and homogeneity of the S1 are particularlyignored while assuming that the σ₁₂ in the wavelength band of the 2-ndharmonics of the YAG laser has such a large absorption cross-section ascan produce a sufficient number of S1 in the case of the Rhodamine 6G.

FIG. 20 illustrates one example of a calculated erase light intensityI(x, y) and a fluorescent intensity F₁(x, y). Here, these individualintensities are standardized with their respective peak values.

As apparent from FIG. 20, the erase light intensity is zero at itscentral portion, and the fluorescent intensity is also high at itscentral portion so that the fluorescent region is left only at thecentral portion. Although, the Rayligh-limit of the condensing opticalsystem is 455 nm, it becomes 100 nm if the half value width of thefluorescent intensity F₁(x, y) is defined as a spatial resolutioncapable of detecting the Rhodamine 6G, thereby exceeding the diffractionlimit of the condensing optical system. In short, it is understood thatthe microscope body of the microscope system of this invention in thepresent example has an excellent super-resolution.

On the other hand, as the YAG laser employed as the light source for thepump light and erase light, an inexpensive, highly safe mode-locked typecan be used. For example, the erase light can be produced by extractinga portion of the light of 2-nd harmonics of 523 nm, which is the pumplight, by the beam splitter and then by converting the wavelength of theextracted portion to 560 nm by a nonlinear crystal such as a highly safeBBO crystal, or a wavelength modulating element such as the Ramanshifter, or a nonlinear medium. Accordingly, the construction of thelight source is remarkably simplified.

When the YAG laser is of the mode-locked type and also thesemiconductor-laser-excited type, it is possible to produce a highlyrepetitive, short pulsed light of 100 MHz and some 10 psecs and to makea small-sized light source of a maintenance-free solid-state.

Moreover, the irradiation light may have a low intensity, and itswavelength is in the vicinity of 500 nm other than the opticalabsorption band of the biological specimen so that a damage on thebiological specimen can be remarkably reduced.

Like the Rhodamine 6G, when 7-ethylamino-4-trifluoromethyl coumarin(C₁₂H₁₀NO₂F₃: Coumarin 500).is used, for example, the pump light and theera se light can be coped with the 2-nd harmonics and the 4-th harmonicsof the YAG laser, as described hereinbefore, so that a spatialresolution of about 100 nm which is the same as that of the Rhodamine 6Gcan be achieved with the erase light intensity of 100 MW/cm².

Unlike the Rhodamine 6G or the 7-ethylamino-4-trifluoromethyl coumarin,when the molecule having the resonance wavelengths λ1 and λ2 which arenot the wavelength formed by modulating the fundamental-wave and theirharmonic-wave of the YAG laser with the simple nonlinear crystal, anoscillation frequency of the YAG laser can be operated in the range fromthe ultraviolet to the infrared waves by modulating with an opticalsystem using a nonlinear crystal such as an optical parametricoscillator (OPO).

Because of the YAG laser, it is quite natural that dye need not beexchanged unlike the existing dye laser so that the operability of themicroscope body is improved.

Example 2

In the foregoing Example 1 the condensed beam of the erase light has thephase distribution in which the phase changes continuously from 0 to 2σwhen turned once around the optical axis. On the contrary, in thepresent Example 2, the condensed beam of the erase light has adiscontinuously changing phase distribution.

In order to give the discontinuously changing phase distribution to thecondensed beam of the erase light, for example, there may be used aphase plate which has a refractive-index distribution (n1˜n8).changingdiscontinuously around the optical axis, as illustrated in FIG. 21.

Here, the intensity distribution of the condensed beam of the eraselight and the intensity distribution of the fluorescence to be emittedare determined in the case where the fluorescence labeler molecule isthe Rhodamine 6G and have the optical parameters and the environmentalparameters as shown in the Table 1 and the Table 2, respectively, andthe condensing optical system of the microscope body is provided with aphase plate having a refractive-index distribution which gives thecondensed beam of the erase light a discontinuous phase distribution ofdiscrete 0, π/2, π and 3π/4 quartered around the optical axis, asillustrated in FIG. 22.

FIGS. 23(a) and (b) show a top plan view and a side elevationillustrating a structure and an optical parameter of the phase plate,respectively.

This phase plate illustrated in FIGS. 23(a) and (b) is formed by coatinga glass substrate (BK-7) with a magnesium fluoride film. This magnesiumfluoride film has a refractive index of 1.38 at a wavelength of 560 nmso that it gives a phase difference of λ/4 for a film thickness of 350nm. Therefore, the individual film thicknesses capable of a phasedistribution of 0, π/2, π and (3π)/2 in the individual quartered regionsaround the optical axis are 350 nm, 700 nm, 1,050 nm and 0 nm,respectively, as illustrated in FIG. 23(a).

The phase plate may be formed not only by making the coating of theoptical thin film such as the magnesium fluoride film having suchrefractive-index distribution but also by etching the glass substratedirectly to make an optical path difference giving the phases of theindividual regions.

Here in the phase plate which is thus formed by coating the glasssubstrate with the optical thin film or by etching the glass substrate,the parallelism or roughness of the glass substrate has to be smallerthan the optical path difference giving the phase difference of π/4, soas to prevent disturbance of the phase plane of the erase light.

In the phase plate using the magnesium fluoride film, more specifically,the disturbance in the optical path difference due to the parallelism orroughness of the glass substrate itself is set to 350 nm or less.

FIG. 24 illustrates the condensed beam intensity of the erase light, ascondensed by the condensing optical system provided with the phase plateof FIG. 23, and the fluorescent intensity.

It is apparent from FIG. 24 that the condensed beam intensity of theerase light takes a shape similar to that of the condensed beamintensity of the erase light illustrated in FIG. 20 of the Example 1,the optical intensity being zero at the central portion.

In other words, an excellent super-resolution is realized even when thecondensed beam of the erase light has the phase distribution changingdiscontinuously around the optical axis.

In addition, it is far simpler to make a phase plate such as a magnesiumfluoride film, for example to have a refractive-index distribution or anoptical-path-difference distribution changing discontinuously around theoptical axis than to make a phase plate to have that changingcontinuously.

Example 3

For the microscope system of this invention, it is the most desirablethat the beam obtained by condensing the erase light that is the lightof the wavelength λ2 be the 1-st-order-Bessel-beam that is anondiffractive beam. However, in order to give the microscope body atheoretical super-resolution, the beam may have the aforementioned shapein which the intensity at the central portion is zero.

Therefore, the beam having a phase modulated by the phase plate, asexemplified in FIG. 18 or 22, may be focused in a reduced scale by theordinary condensing optical system, i.e., the condensing optical systemnot having the zonal optical system as in the Example 1. Then, althoughnondiffraction disappears, an excellent super-resolution can berealized.

FIG. 25 illustrates an intensity distribution of a condensed beam of anerase light and a fluorescent intensity distribution, which arecalculated when the specimen is dyed with the rhodamine 6G and theshading ratio of the pupil is 0 and the optical parameters and theenvironmental parameters a takes the values of the Table 1 and the Table2, respectively.

It is apparent from FIG. 25 that the beam condensed by the ordinarycondensing optical system having the phase plate is shaped to have azero intensity at its central portion, thereby leaving the fluorescentregion at its central portion. The Raileigh-limit of the condensingoptical system is 455 nm but becomes 200 nm exceeding the diffractionlimit of the condensing optical system if the half value width of thefluorescent intensity F₁(x, y) is the spatial resolution capable ofdetecting the Rhodamine 6G. It is, therefore, understood that anexcellent super-resolution is achieved.

Example 4

FIG. 26 illustrates one example of the microscope system of thisinvention having a super-resolution microscope function.

In this microscope system illustrated in FIG. 26, an adjusted specimen(100) is dyed with the Rhodamine 6G used as the fluorescence labelermolecule.

There are provided a mode-locked type Nd:YAG laser (1) as the lightsources for the pump light which is the light of the wavelength λ1 andthe erase light which is the light of the wavelength λ2 and a BBOcrystal (2).which is a nonlinear crystal as the nonlinear medium fortheir wavelength conversions. The fundamental-wave of the Nd:YAG laser(1) is subjected to a wavelength conversion with the BBO crystal (2),thereby oscillating the 2-nd-harmonics of 532 nm as the pump light.

On the optical path of this pump light, there is provided a half mirror(3), by which a portion of the 2-nd harmonics, i.e., the pump light isextracted and subjected to a wavelength conversion to 563 nm by a Ramanshifter (4) made of a Ba(NO₃)₂ crystal as the nonlinear crystal therebyto produce the erase light.

This erase light irradiates a phase plate (6) of the Example 2 through amirror (5) so that it is formed by the phase plate (6) into a hollowbeam having a zero field intensity at its central portion.

The erase light and the pump light thus formed as the hollow beam arecaused by a dichroic mirror (7) to follow the same optical path and arecondensed through a next dichroic mirror (8) and a condensing objectivelens (9) on the adjusted specimen (100) which is carried on atwo-dimensional carriage stage (10) moving in the arrow direction asshown.

By the irradiations of the pump light and the erase light thuscondensed, the fluorescence emitted from the adjusted specimen (100) isreflected by the dichroic mirror (8) and is condensed on aphotomultiplier (14) by a fluorescence condenser lens (11) through asharp cut filter (12) and a pin hole (13).

The dichroic mirror (8) is an interference filter capable oftransmitting the pump light and the erase light and having areflectivity in the fluorescent band. As a result, the pump light andthe erase light can be separated from the fluorescence that is a signallight.

The sharp cut filter (12) arranged between the dichroic mirror (8) andthe photomultiplier (14) is a band-pass filter for cutting the pumplight and the erase light which are moved by surface scatter of thedichroic mirror (8) and the like, and the pin hole (13) functions as aspatial filter for cutting the stray light which is spatially diffused.By these sharp cut filter (12) and pin hole (13), the detectionsensitivity of the fluorescence and the S/N ratio are improved.

In this microscope system of this invention, the intensity of thefluorescence can be monitored while moving the two-dimensional carriagestage (10) synchronously with the timing of irradiation of the pulselight of the Nd:YAG laser, thereby to produce a two-dimensionalfluorescent image of the adjusted specimen (100).

Here, the intensities of the pump light and the erase light aremonitored by a photomultiplier (15) so that the fluctuation of thegraphic signal for each pixel due to the intensity conversion of thelaser beam can be inhibited by adding the signal processing thereby toimprove the image quality.

In this example shown in FIG. 26, each component can certainly bemodified in various manners and another function can be added.

For example, if the ordinary optical lens is used as the condensingobjective lens (9), it is possible to form a condensed beam of the eraselight having a zero intensity at the center of the condensed point.Further, if the condensing objective lens (9) gives a boundary conditionsymmetric around the optical axis, it is possible to form a1-st-order-Bessel-beam that is a nondiffractive beam.

Hereinafter, examples of an optical system as the condensing objectivelens (9) capable of forming an 1-st-order-Bessel-beam are described.

(I) Zonal Optical System

This zonal optical system may be exemplified by combining an annularzonal slit (16) and an ordinary glass lens (17), as illustrated in FIG.27. When the annular zonal slit (16) is employed for example, it isdesireble to place the etalon(not-shown).in front of the zonal slit(16), thereby to increase the quantity of light passing therethrough byits one-dimensional diffractive light.

On the other hand, there is a zonal optical system intrinsicallyequipped with a zonal-pupil, which has a boundary condition necessaryfor forming the 1-st-order-Bessel-beam. This zonal optical system may beexemplified by a reflecting objective lens illustrated in FIG. 28. Thisreflecting objective lens of FIG. 28 is the Cassegrain or Schwaltshildtype optical system, in which a convex reflecting mirror placed insideshades the central portion of the optical pupil so that it substantiallyplays a role similar to that of the aforementioned annular zonal slit(16). The reflecting type optical system is also exemplified by a Walterlens of the oblique-incident type, as illustrated in FIG. 29. ThisWalter lens is an extreme zonal optical system and is equivalent to theuse of a substantially ideal annular zonal slit.

(II) Diffractive Optical System

An axially symmetric diffractive optical system (including thetransmission type and the reflection type) gives a boundary conditionsymmetric around the optical axis to the aforementioned wave equation 12so that it can be applied as the condensing optical system.

This diffractive optical system may be exemplified by a Fresnel zoneplate. FIGS. 30(a) and 30(b), respectively, illustratestransmission-type Fresnel zone plate and a reflection-type Fresnel zoneplate. Since the Fresnel zone plate intrinsically has a focusingability, it has both a condensing ability and a boundary conditionnecessary for forming the 1-st-order-Bessel-beam.

When it is intended to give only the boundary condition, on the otherhand, there may be provided a diffraction grating which has grooves orspiral grooves in concentric circles, as illustrated in FIG. 31.

(III) Axicon

This axicon is also an axially symmetric 6optical system capable offocusing a-point light source on an axis over a wide range on the axis.FIG. 32 illustrates one example of the axicon. This axicon exemplifiedin FIG. 32 is a reflecting lens called the “McLeod” which has a conicalsurface and a plated flat surface. In this axicon, for a point on anaxis within a predetermined range, there exists an identical point onthe axis, and, for a point out of an axis, there exists an image pointat a point symmetric with that point. This axicon also gives a boundarycondition symmetric to the optical axis.

The optical system thus far described can be provided as the condensingobjective lens (9).

In the example shown in FIG. 26, on the other hand, there may beprovided as the fluorescence detector not only a photoelectronmultiplier such as the photomultiplier (14) (15) but also asemiconductor detector such as a PIN photodiode or a CCD.

When the Rhodamine 6G is used as the fluorescence labeler molecule,another means for the light source can be employed. For example, inorder to produce the erase light, the Raman shifter (4) can be replacedby an OPO or a dye laser. In this case, the erase light has a variablewavelength, so that the rhodamine group molecule such as Rhodamine 110can be used as the labeler molecule.

Moreover, in the microscope system of FIG. 26, in order to produce atwo-dimensional fluorescent image of the adjusted specimen (100), thetwo-dimensional carriage stage (10) is capable of movingtwo-dimensionally with respect to the condensing beam. In addition, asin the laser scanning type microscope of the prior art, for example, theoptical system can be vibrated directly with a galvan-mirror and thelike, thereby making it possible to scan the adjusted specimen (100)two-dimensionally with the beam.

Example 5

FIG. 33 illustrates one example of the microscope system of thisinvention having a confocal type super-resolution microscope function.

In this microscope system of FIG. 33, the adjusted specimen (100) isdyed with the Coumarin 500.

As the light sources for the pump light and the erase light, there isprovided the mode-locked type Nd:YAG laser (1), and the fundamental-wave(1,064 nm).of this Nd:YAG laser (1) is diverged by a half mirror (28).

The fundamental-wave of one path is subjected to a wavelength conversionto 355 nm by a 3-rd harmonics generator (18) made of BBO-1 crystal,thereby making it into the pump light. The fundamental-wave of anotherpath is introduced through a mirror (29) into a 2-nd harmonicsgenerator. (19) made of BBO-2 crystal, by which it is subjected to awavelength conversion to 532 nm, thereby making it into the erase light.

The erase light is guided by a mirror (30) to irradiate a phase plate(31), as exemplified in the Example 2, by which it is shaped to have azero field intensity at its central portion.

Then, the erase light thus shaped into the hollow beam and the pumplight are guided to follow the same optical path by a dichroic mirror(20).

Between the dichroic mirror (20) and the 3-rd harmonics generator (18),there is arranged a polarizer (21), by which the polarizing plane of thepump light can be freely rotated.

The pump light and the erase light, as arranged to have their opticalpaths on the common axis by the dichroic mirror (20), are guided toilluminate a pin hole (23) by a condenser lens (22) which is theSchwaltshild type reflecting optical system.

The image of the pin hole (23) illuminated with the condensed pump lightand erase light is employed as the light source for forming a microbeam.

The pump light and erase light having passed through the pin hole (23)penetrate a dichroic mirror (24) and then are condensed by an objectivelens (25) which is the Schwaltshild type reflecting optical system onthe adjusted specimen (100) placed on the two-dimensional carriage stage(10).

The condenser lens (22) and the objective lens (25), i.e., theSchwaltshild type reflecting optical system have each mirror surfacecoated with a metal film such as aluminum and can focus a light of awide wavelength range from infrared to ultraviolet without any chromaticaberration. As a result, they can focus the pump light and the eraselight having different wavelengths on the adjusted specimen (100) inabsolutely the same focusing performance and high resolution. ThisSchwaltshild type reflecting optical system is a zonal optical systemand gives a boundary condition necessary for forming the1-st-order-Bessel-beam when a shading ratio ρ₀ falls within a range from0 to 1 by adjusting radius of its convex mirror placed inside Thefluorescence emitted from the adjusted specimen (100) by irradiation ofthe condensed pump light and erase light It condensed is reflectedthrough the objective lens (25) by the dichroic mirror (24). At thistime, the scattered light and the stray light of the pump light and theerase light are not reflected by the dichroic mirror (24) so that onlythe fluorescent light that is the signal light can be separated.

Then, the fluorescent light reflected by the dichroic mirror (24) passesthrough a pin hole (26), and after the afterglows of the pump light andthe erase light are cut by a sharp cut filter (27), it is condensed onthe receiving surface of the photomultiplier (14).

This optical system of the microscope body in the microscope system ofthis invention, as illustrated in FIG. 33, is a confocal optical system,in which the pin holes (23) and (26) are located optically at theconfocal position with the condensing point on the adjusted specimen(100) being a center. Hence, like the scanning-laser fluorescentmicroscope having a similar confocal optical system, it is possible toachieve an excellent S/N ratio and further, by the movement of thetwo-dimensional carriage stage (10) indirection of the optical axis, toproduce a three-dimensional fluorescent image of the adjusted specimen(100) can be produced in an excellent S/N ratio.

In addition, by the polarizer (21), a new useful function is added tothe super-resolution function. Generally, a molecule has an intenseabsorption for an electric vector in a specific direction, and thebenzene derivative or the purine derivative, for example, absorbs alight having an electric vector in the same direction as the planardirection of the molecule plane. In this case, by turning thepolarization direction of the pump light, it is possible to excite onlythe molecule spatially oriented in a specific direction, thereby toestablish fluorescence. As a result, by taking a fluorescent image whilechanging the polarization direction of the pump light by the polarizer(21), spatial orientation characteristics of a specific molecule orstructures of the adjusted specimen (100) can be analyzed.

Example 6

FIG. 34 illustrates another example of the microscope system of thisinvention.

The microscope system of FIG. 34 has not only the super-microscopefunction but also a micro-manipulator function using a hollow microbeam. The microscope system further has the ordinary fluorescentmicroscope function so that it can always monitor the fluorescent imagefrom the adjusted specimen (100) on the real time without any laserscanning.

In this microscope system of FIG. 34, the adjusted specimen (100) isdyed with the Rhodamine 6G.

As a light source for the pump light and the erase light for thesuper-resolution microscope function and as a nonlinear medium forwavelength conversion, there are provided a mode-locked type Nd:YAGlaser (32) and a KTP crystal (35) or the nonlinear crystal,respectively. As a laser light source for forming a hollow micro beamfor the micro manipulator function and as a nonlinear medium forwavelength conversion, there are provided a Nd:YAG laser (33) ofcontinuous oscillation CW and axiconTP crystal (36), respectively.Further, as a light source for the ordinary fluorescent microscopefunction, there is provided a mercury lamp (31).

First, here will be described the super-resolution microscope function.

The fundamental-wave of the Nd:YAG laser (32) are subjected to thewavelength conversion by the KTP crystal (35), so that the 2-ndharmonics of 532 nm are oscillated as the pump light. Part of this pumplight is extracted by a half mirror (37) to go through a mirror (38)into a Raman shifter (39) made of Ba(NO₃) crystal, then its wavelengthis changed to 563 nm by the Raman shifter (39), thereby to produce theerase light.

Its The erase light goes into a dichroic mirror (40), by which the 2-ndharmonics of 532 nm having contaminated are removed, so that the onlythe light of 563 nm is extracted in a high purity.

This erase light is further shaped into the hollow beam having a zerofield intensity at its central portion by the phase plate (6), such asthe one exemplified in the Example 2.

These erase light and pump light thus shaped into the hollow beam arecaused to pass through the same optical path by a dichroic mirror (41).

Between the half mirror (37) and the dichroic mirror (41), there isarranged the polarizer (21), by which the polarizing plane of the pumplight can be freely rotated.

The pump light and the erase light, as arranged to have their opticalpaths on the common axis by the dichroic mirror (41), are shaped by arelay lens (42) and are reflected by a half mirror (43) to go into theobjective lens (9), by which they are condensed on the adjusted specimen(100) carried on the two-dimensional carriage stage (10).

The fluorescence emitted from the adjusted specimen (100) by theirradiations of the pump light and the erase light passes through thehalf mirror (43) and a half mirror (44) and is reflected in a directionto go into a lens (46) by a half mirror (45). Then, the fluorescence iscondensed at the center of a pin hole (47) by the lens (46) and goesthrough a lens (48) into a spectrometer (49).

The pin hole (47) functions as a spatial filter and plays a role toenhance S/N ratio for the measurements by cutting, for example, thefluorescence which is emitted from such as an optical system, other thanthe adjusted specimen (100).

In this example shown in FIG. 34, on the other hand, a spectrometer (49)is provided as a fluorescence detector thereby to make it possible notonly to measure fluorescent intensity but also to observe fluorescencespectrum and measure time response to laser irradiation thereby toanalyze chemical structure or composition of the adjusted specimen(100). Moreover, spatial orientation data of the composition can beobtained by changing the polarization planes of the pump light and theerase light relatively by the polarizer (7).

Thus, there is achieved a remarkably excellent super-resolutionmicroscope function capable of making various measurements and analysesfor the adjusted specimen (100).

Next, the micro manipulation function will be described.

When particles are to be captured in the micro manipulation, it isbasically necessary to use a continuously oscillating light source. Forthis necessity, the Nd:YAG laser (33) of CW is provided as thecontinuously oscillating light source.

The fundamental-wave of this Nd:YAG laser (33) are subjected to awavelength conversion by the KTP crystal (36) to produce 2-nd harmonicsof 532 nm. The 2-nd harmonics are employed as a light source forgenerating the hollow micro beam to be used for the micro manipulation.

The 2-nd harmonics having passed through the half mirror (38) aresubjected to a wavelength conversion to 563 nm by the Raman shifter(39), and the 2-nd harmonics of 532 nm having contaminated are removedby the dichroic mirror (40), so that only the light of 563 nm isextracted with high purity.

This light of 563 nm is shaped by the phase plate (6) to the hollowmicro beam having a zero field intensity at its central portion, andpasses through the dichroic mirror (41) and the relay lens (42), and isreflected by the half mirror (43) to go into the condensing objectivelens (9), by which it is condensed on the adjusted specimen (100).

Thus, the hollow micro beam of 563 nm for the micro manipulation canemploy the same optical system as the aforementioned one for the eraselight for the super-resolution function.

Although the micro manipulation of the prior art, as called the “opticalpincette”, can only move a single particle, this micro manipulationusing the hollow micro beam can confine a plurality of particles in thehollow micro beam and can function as the “optical pipette” for a highgrade micro manipulation. Of course, the laser intensity can be aremarkably low level thus the damage on the specimen can be lowered.

The micro manipulation is performed while monitoring the entiremicroscope image. Thus, there is added the ordinary fluorescentmicroscope function for monitoring the microscope image.

As the light source for this fluorescent microscope function, there isprovided the mercury lamp (31), and the light therefrom is guidedthrough the half mirrors (44) and (43) and the condensing objective lens(9) to irradiate the adjusted specimen (100). Then, the fluorescentimage emitted by that light irradiation is guided again through thecondensing objective lens (9), and the half mirrors (43) and (44) to gointo a CCD cameral focusing lens (50), by which it is directly focusedon the receiving surface of a CCD camera (51). This fluorescent imagecan be directly monitored at any time on the CRT.

Accordingly, while monitoring the fluorescent image on the CRT,particles such as red blood cells can be micro-manipulated at a highgrade.

Example 7

In the microscope system of this invention, in order to give anexcellent super-resolution and operability to its microscope body, itsis desirable that the beam obtained by condensing the erase light be the1-st-order-Bessel-beam.

The 1-st-order-Bessel-beam can be formed by using the zonal opticalsystem, the diffractive optical system and the axicon, or the phaseplate of the condensing optical system,as described hereinbefore. Inaddition, by giving the boundary condition necessary for forming the1-st-order-Bessel-beam into the resonator of a gas laser, a sold laseror a semiconductor laser of the light source for the erase light, it ispossible to make the erase light itself into the 1-st-order-Bessel-beam.

FIG. 35 illustrates one example of a laser resonator provided with theboundary condition.

This laser resonator illustrated in FIG. 35 is provided with a lens (53)having an ordinary focal distance f, a phase plate (54) and an outputmirror (55) and further with a ring-shaped zonal mirror (52) as aresonator mirror on the end face.

The 1-st-order-Bessel-beam can be directly produced by giving theboundary condition axially symmetric with respect to the beam opticalaxis in the ring-shaped zonal mirror (52) and by placing in the laserresonator the phase plate which gives the beam the phase difference inwhich the electric fields axially symmetric with respect to the electricfield of the plane normal to the optical axis are shifted by π from eachother.

By placing such ring-shaped zonal mirror (52) or phase plate in thelaser resonator to give the boundary condition, the structure of thecondensing optical system of the microscope body can be simplified,thereby remarkably simplifing its alignment or the like.

FIG. 36 illustrates one example of the microscope system of thisinvention when a Nd:YAG laser (56) has a laser resonator having theconstruction of FIG. 35.

In this example shown in FIG. 36, the adjusted specimen (100) is dyedwith the Rhodamine 6G, and there is provided as the light source themode-locked type Nd:YAG laser (56) which has a laser resonator of theconstruction of FIG. 35.

This Nd:YAG laser (56) can produce the fundamental-wave of the1-st-order-Bessel-beam directly. The pump light is produced byconverting the wavelength of the fundamental-wave into 2-nd harmonics of532 nm by axiconTP crystal (57), and the erase light is produced byconverting the wavelength of a portion of the 2-nd harmonics to 563 nmby the Raman shifter (4) made of a Ba(NO₃)₂ crystal.

Since this erase light is already the 1-st-order-Bessel-beam, thecondensing optical system need not be provided, as in the foregoingembodiments, with the phase plate (6) for converting the erase lightinto the hollow beam or the Al optical system such as the zonal opticalsystem for converting the same into the 1-st-order-Bessel-beam, thus theerase light can be condensed direcly on the adjusted specimen (100)through the dichroic mirrors (7) and (8) and the condensing objectivelens (9).

Of course, the condensed beam on the adjusted specimen (100) is the1-st-order-Bessel-beam.

Since the optical system for forming the 1-st-order-Bessel-beam is thusfirmly assembled in the laser, the condensing optical system can have asimple construction but can be strong against dislocation and excellentin stability to improve the super-resolution and operability better.

As the optical system capable of giving the boundary condition forforming the 1-st-order-Bessel-beam, not only the aforementionedring-shaped zonal mirror (52) or the phase plate for giving the beam thephase difference in which the electric fields axially symmetric withrespect to the electric field of the plane normal to the optical axisbut also the zonal diffraction grating, the Fresnel zone plate or thezonal aperture and the like can be placed in the resonator of the laserlight source for the erase light.

Generally, the laser can generate beam patterns having various modepatterns in dependence upon the construction of its resonator. In thevibrational mode of a higher order of the Gauss's type, Laguerre's typeor Hermitian's type, therefore, there exists a pattern in which theintensity is zero at the central portion of the laser beam, asillustrated in FIGS. 37(a) to 37(d).

Hence, the various lasers of the light source can have an excellentsuper-resolution, as the 1-st-order-Bessel-beam has, not only by givingthe laser resonator the construction illustrated in FIG. 35, but also byoscillating the laser beam having the vibrational modes of higher ordersof the Gauss's type, Laguerre's type or Hermitian's type.

Example 8

In the microscope system of this invention thus far described, the S/Nratio of the fluorescence signal to go into the emission detector fromthe specimen can be improved by using several filter elements.

In the super-resolution microscope, generally, there exist not only thefluorescence from the observatory specimen to be detected but alsoseveral background lights. These background lights are: i) the scatteredlight of the pump light; ii) the scattered light of the erase light; andiii) the fluorescence from the optical system other than the specimen.

i) This scattered light has the wavelength λ1 of the pump light and isscattered mainly on the surface of the condenser lens or at the boundaryof the cover glass for protecting the observatory specimen.

ii) This scattered light has the wavelength λ1 of the erase light forthe same reason as that of i). The erase light has a higher intensitythan that of the pump light so that it raises an obstruction to thefluorescence detection.

iii) If the glass material for the lens or cover glass of the opticalsystem is an ideal non-fluorescent quartz or a fluorite, there exists nofluorescence having a wavelength range of 250 nm or more. In a poorglass material, however, a fluorescence comes from the impure portion orcolor center.

These background lights may also be produced in the super-resolutionmicroscope body of the microscope system of this invention. Therefore,the background lights are desirably prevented from migrating into thefluorescence and going into the detector, thereby to improve the S/Nratio.

As illustrated in FIG. 38, basically, the background lights can becompletely eliminated by a combination of the optical filter and thespatial filter.

Ordinarily, the wavelength λ1 of the pump light is the shortest, and thewavelength λ2 of the erase light and the wavelength band of thefluorescence at the deexcitation from S1 to S0 on the longer wave side.

In the Rhodamine 6G, the absorption band from S1 to S2 and thefluorescence wavelength band from S1 to S0 overlap each other, and thewavelength λ2 and the fluorescence wavelength to be detected are closeto each other. When such molecule is to be used as the fluorescencelabeler molecule, therefore, the background lights are desired to becarefully eliminated.

First of all, the scattered light i) of the pump light can beeliminated, because the wavelengths of the pump light and the eraselight are generally apart from each other, by providing a sharp cutfilter which is prepared by diffusing a absorbent containing a polymerover the glass substrate or coating the glass substrate with aninterference film.

It is apparent from FIG. 38, for example, that the shorter wave sidethan the wavelength λ2 can be completely eliminated by such sharp cutfilter. For example, if the sharp cut filter using a dielectricmulti-layer film is designed for the optimum, giving an interval ofabout±30 nm of the wavelength-separating-design position, the light onthe shorter wave side of the wavelength width can be eliminatedsubstantially by 100% whereas the light on the longer wave side can betransmitted. In the Rhodamine 6G, since the fluorescence measurementwavelength region and the pump light are apart from each other by 40 nmor more, as has been illustrated in FIG. 14, the scattered light of thepump light can be separated and eliminated from the fluorescence comingfrom the specimen by the sharp cut filter.

This sharp cut filter can be placed on the optical path of thefluorescence in front of the fluorescence detector (or thephotomultiplier (14)), as illustrated in FIG. 26 of the foregoingExample 4.

Next, the erase light ii) can be eliminated by providing a notch filter.The notch filter is one using a dielectric multi-layered film not totransmit only a predetermined wavelength, as illustrated in FIG. 38. Thelight in a band of about 20 nm around the designed wavelength can becompletely eliminated if more of the dielectric multi-layered film arelaminated to optimize thickness of the dielectric multi-layered film.

Especially in the case of the Rhodamine 6G, the fluorescence emissionregion extends to 550 to 650 nm, and the wavelength of the erase lightis 562 nm. By the notch filter, however, the scattered light of theerase light in the vicinity of 562 nm can be prevented from entering thedetector.

On the other hand, when the emission of the fluorescence from thespecimen is inhibited as in the Rhodamine 6G by making use of the doubleresonance absorption process and the induced emission, the absorptionband from S1 to S2 and the fluorescence wavelength band from S1 to S0overlap each other, so that the fluorescence going into the emissiondetector from the specimen is partially lost. By using the notch filter,however, only the fluorescence in a band of about 20 nm including 562 nmof the region of 550 to 650 nm is lost, so that the loss of thefluorescence to be observed can be minimized.

Furthermore, in order to eliminate the background light other than thefluorescence from the specimen more completely, it is also preferable touse a band-pass filter. This band-pass filter is prepared by coating theglass substrate with a dielectric multi-layered film and, on thecontrary to the notch filter, contains a specific wavelength to transmitonly the light of the wave region around that specific wavelength.

Consequently, with the band-pass filter not transmitting the wavelengthsof the pump light and the erase light but transmitting only thefluorescence emission wavelength band, the wavelengths other than thatof the fluorescence from the specimen can be completely cut.

As described hereinbefore, by providing the emission condensing opticalsystem for condensing the emission from the specimen to the emissiondetector, for example, with the sharp cut filter, the notch filter andthe band-pass filter, the fluorescence to be emitted when thefluorescence labeler molecule is to be deexcited can be detected at aremarkably excellent S/N ratio.

For the elimination of the fluorescence from the optical system otherthan the specimen iii), since the fluorescence is caused by the impurityor the color center of the glass material of the lens of the opticalsystem or the cover glass, no problem arises if an ideal syntheticquarts (i.e., non-fluorescent quarts) or a fluorite is utilized.

Such glass material may be exemplified not only by the synthetic quartsbut also by CaF₂, NaF, Na₃AlF₆, LiF, MgF₂, SiO₂, LaF₃, NdF₃, Al₂O₃,CeF₃, PbF₂, Mgo, ThO₂, SnO₂, La₂O₃ or SiO.

Of course, it is desired to provide a filter optical system forseparating the fluorescence coming from the specimen and thefluorescence coming from the optical system other than the specimen.Such filter optical system may be exemplified by a spatial filter suchas a slit or a pin hole.

FIG. 39 illustrates one example of the microscope system of thisinvention, which is provided with the filter optical system.

In the filter optical system of this microscope system of FIG. 39, thereare arranged, in a shade box (62) and in front of the notch filter (14),a notch filter (58), a band-pass filter (59) and a sharp cut filter (60)in the recited order. Moreover, a pin hole (61) is formed in the facialportion of the shade box (62) facing the dichroic mirror (24).

On the other hand, the side of the adjusted specimen (100) to which thelaser beam is irradiated is covered with and protected by a cover glass(63). This cover glass (63) is formed of the aforementioned glassmaterial, for example.

In this microscope system of FIG. 39, the pin hole (23) and the pin hole(61) functioning as a spatial filter take confocal positions withrespect to an objective lens (64) and the face of the adjusted specimen(100). Although apparent from a ray tracing in the case of such confocaloptical system, the fluorescence emitted from other than the focal pointof the pump light and the erase light, i.e., other than the specimenface cannot pass through the pin hole (61) and cannot reach thereceiving face of the photomultiplier (14) that is an emission detector.

For example, the fluorescence emitted from the cover glass (63) is notfocused on the pin hole (61) if it passes through the objective lens(64). As a result, the unfocused numerous fluorescences cannot passthrough the pin hole (61). On the other hand, the fluorescence emittedfrom the face of the objective lens (64) is not condensed directly onthe pin hole (61) by the objective lens (64) so that it does not passthrough the pin hole (61) or reach the receiving face of thephotomultiplier (4).

According to the notations of FIG. 38, the fluorescence intensityI_(signal) going into the emission detector can be expressed by thefollowing equation:

I _(signal)=∫_(λ) _(ex1) ^(λ) ^(_(ex2)) I(λ)dλ=∫_(λ) _(ex1) ^(λ)^(_(ex2)) T ₁(λ)T ₂(λ)T₃(λ)F(λ)dλ  Equation 19

In this equation, F(λk) indicates a fluorescent intensity of thefluorescence labeler molecule, and λ_(ex1) and λ_(ex2) indicate thelower limit wavelength and the upper limit wavelength of the sensitivityrange of the emission detector, respectively. As shown in FIG. 38,I_(signal) corresponds to the fluorescent intensity from the specimenwithout migration of the background lights.

Here in FIG. 39, if the objective lens (64) is a reflecting objectivelens, there is no fluorescence from the lens glass material so that theSIN ratio can be better improved.

Example 9

FIG. 40 shows one example of an electric system corresponding to themicroscope system of this invention, as illustrated in FIG. 34 ofExample 6.

In the example shown in FIG. 40, all the systems in the microscopesystem of this invention are controlled basically by a personal computer(66).

This personal computer (66) controls the oscillations of the YAG laserand the drive of the two-dimensional carriage stage (10) that is ascanning stage of the adjusted specimen (100) of FIG. 34, for example.

The timings of the system are all based on the clock of the personalcomputer (66). This clock is divided by a frequency divider (67) into afrequency capable of oscillating a laser, and the clock signal thusdivided becomes a Q-switch signal and a flash lamp signal for the lasercontrol by delay and waveform shape with a gate & delay generator (68)to control the YAG laser.

The fluorescent spectrum at each laser shot is monitored by a CCD array(69). More specifically, in response to the laser shot, morespecifically, the fluorescence emitted from the adjusted specimen (100)is divided by a diffraction grating (70) and then is detected as afluorescent spectrum by the one-dimensional CCD array (69).

The stored data of each pixel of the CCD array (69) are transferred ateach laser shot to the memory of the personal computer (66) while beingsynchronized with the movement of the two-dimensional carriage stage(10) and the laser emission.

From the fluorescent spectral data stored in the memory of the personalcomputer (66), only the data of a predetermined fluorescent wavelengthare extracted by the numerical operations of the personal computer (66),and with this extracted data, a two-dimensional scanned image of theadjusted specimen (100) is formed.

By analyzing a two-dimensional scanned image graphically is for eachmeasured wavelength, it is possible not only to achieve a merefluorescent image but also to analyze the two-dimensional composition.

Moreover, the fluorescent image of the adjusted specimen (100), asobtained by the irradiation of the mercury lamp (31), is simultaneouslymonitored by a CCD camera (71), and its fluorescent image data can bestored in a frame memory (72) at any time.

As a result, separately of the two-dimensional scanned image, the entirefluorescent image of the adjusted specimen (100) can be monitored at anytime. This function is remarkably convenient especially for the micromanipulation using the hollow micro beam, as has been describedhereinbefore.

In addition, the personal computer (66) can control a CRT (73) and theframe memory (72) to display and process the image whenever necessary.The graphic data thus prepared can be outputted by the CRT (73) or avideo printer (74), for example.

This invention should not be limited to the foregoing Examples but cantake various modes in detail.

INDUSTRIAL APPLICABILITY

According to this invention, as has been described in detailhereinbefore, there is provided a novel microscope system which iscapable to condense, in an excellent beam profile, an erase lightexciting a molecule in the first excited state to the second excitedstate by using a simple, compact optical system and which has highstability and operability and an excellent super-resolution. Alsoprovided is a novel microscope system which has a micro manipulatorfunction to capture and move specimen particles, without damaging thespecimen, by using the erase light being a hollow beam.

FIGS. 1 to 4

Valence Orbit 4 (Vacant Orbit)

Valence Orbit 3 (Vacant Orbit)

Valence Orbit 2

Valence Orbit 1

Inner-shell Orbit

Occupant Electron

Wavelength λ1

Wavelength λ2

Fluorescence or

Phosphorescence

FIG. 5

Second Excited State

First Excited State

Ground State

FIG. 6

Fluorescence Disappearing Area A1

Fluorescence Area A0

Second Excited State

First Excited State

Ground State

Observation Area

FIGS. 7 to 11

λ1 Pulse Beam

Molecule No.

in First Excited State

(Fluorescent Intensity)

without

λ2 Pulse Beam Irradiation

λ2 Pulse Beam

Molecule No.

in First Excited State

(Fluorescent Intensity)

with

λ2 Pulse Beam Irradiation

Time Axis

Gate Pulse

Measurement Time

FIG. 12

Vibrational Relaxation

Vibrational Relaxation

Vibrational Relaxation

Vibrational Relaxation

Internal Conversion

Internal Conversion

Inter-System Crossing

Light Absorption

Fluorescence

Phosphorescence

FIG. 14

Cross-section

Wave Number

Wavelength (nm)

Harmonic Curve

Rhodamine6G

(Ethanol Solution)

FIG. 19

Pupil

where

Maximum Transmittance of Objective

Maximum Transmittance of Multilayer

Shading Ratio

FIGS. 20, 24 & 25

Standardized Fluorescence / Erase Light Intensity

(Arbitrary Unit)

Spatial Distance

(micron)

Erase Light

Intensity

Fluorescence

Intensity

Erase Light

Wavelength: 560 nm

Laser Light

Pulse Width: 150 psec

Maximum Intensity

of Erase Light

:36 MW/cm²

Aperture Ratio

:100%

FIG. 23

Magnesium Fluoride Film

Glass Substrate

FIG. 26

Double Wave

Raman Shifter

FIG. 27

Object Point

Condense Point

FIG. 28

Cassegrain (Schwalzschild) Type

Reflecting Mirror

FIG. 29

Walter Type

FIG. 30

Under-Film

FIG. 31

AlGaAs Upper Cladding

GaAs Quantum Well

AlGaAs Core

AlGaAs Lower Cladding

GaAs Substrate

FIG. 32

Reflecting Surface

Microscope

Lamp

Mirror with Pinhole

FIG. 35

Output

FIG. 36

Double Wave

Raman Shifter

FIG. 38

Cross-section

Wavelength (λ)

Wavelength (λ)

Wavelength (λ)

Wavelength (λ)

Wavelength (λ)

Absorption Band

Absorption Band

Fluorescent Emission Band

Sharp Cut

Filter

Transmittance

Band-Pass

Filter

Transmittance

Filter

Transmittance

Detector

Input Signal

FIG. 40

66 Personal Computer

67 Frequency Divider

68 Gate & Delay Generator

69 CCD Array

70 Diffraction Grating Fluorescence (by Laser Irradiation)

71 CCD Camera Fluorescent image (by mercury lamp)

72 Frame memory

74 Video Printer (Q-switch) flashlamp

What is claimed is:
 1. A microscope system comprising: an adjustedspecimen; and a microscope body; wherein said adjusted specimen is dyedwith a molecule which has three electron states including at least aground state and which has an excited wavelength band from a firstelectron excited state to a second electron excited state which overlapsa fluorescent wavelength band upon deexcitation through a fluorescenceprocess from the first electron excited state to a vibrational level inthe ground state; wherein said microscope body includes: a light sourceoperable to provide light having a wavelength λ1 for exciting themolecule from the ground state to the first electron excited state; alight source operable to provide light having a wavelength λ2 forexciting the molecule in the first electron excited state to the secondor higher electron excited state; a condensing optical system operableto condense the light having the wavelength λ1 and the light having thewavelength λ2 on said adjusted specimen; an overlap device operable topartially overlap an irradiation region of the light having thewavelength λ1 and an irradiation region of the light having thewavelength λ2 on said adjusted specimen; and an emission detectoroperable to detect an emission upon deexcitation of the excited moleculeto the ground state; wherein a region of the emission upon deexcitationof the molecule from the first electron excited state to the groundstate is inhibited by irradiating the light having the wavelength λ1 andthe light having the wavelength λ2 through said overlap device; andwherein a beam obtained by condensing the light having the wavelength λ2has a phase distribution in which the phase is shifted by π at asymmetric position with respect to an optical axis of the beam in aplane normal to the optical axis.
 2. A microscope system of claim 1,wherein the beam obtained by condensing the light having the wavelengthλ2 has a phase distribution in which the phase changes continuously from0 to 2π when turned once around the optical axis in a plane normal tothe optical axis.
 3. A microscope system of claim 1, wherein anexcitation wavelength band from the first electron excited state to thesecond electron excited state and an excitation wavelength band from theground state to the first electron excited state are different.
 4. Amicroscope system of claim 1, wherein an optical axis of a beam obtainedby condensing the light having the wavelength λ1 and the optical axis ofthe beam obtained by condensing the light having the wavelength λ2 arecoaxial.
 5. A microscope system of claim 1, wherein the beam obtained bycondensing the light having the wavelength λ2 has a phase distributionin which the phase changes discontinuously from 0 to 2π when turned oncearound the optical axis in the plane normal to the optical axis.
 6. Amicroscope system of claim 1, wherein the beam obtained by condensingthe light having the wavelength λ2 is a Bessel beam.
 7. A microscopesystem of claim 6, wherein the Bessel beam is a 1-st-order-Bessel-beam.8. A microscope system of claim 1, wherein the beam obtained bycondensing the light having the wavelength λ2 is a laser beam having avibrational mode of any one of a Gauss's type, Laguerre's type andHermitian's type.
 9. A microscope system of claim 1, wherein any of agas laser, a solid laser and a semiconductor laser is provided as saidlight source for the light having the wavelength λ1.
 10. A microscopesystem of claim 9, wherein an oscillation wavelength of any of the gaslaser, the solid laser and the semiconductor laser is the wavelength λ1.11. A microscope system of claim 9, wherein a harmonic-wave of anoscillation wavelength of any of the gas laser, the solid laser and thesemiconductor laser has the wavelength λ1.
 12. A microscope system ofclaim 9, wherein a sum frequency of, or a difference frequency between,an oscillation wavelength of any of the gas laser, the solid laser andthe semiconductor laser and a harmonic-wave of the oscillationwavelength has the wavelength λ1.
 13. A microscope system of claim 9,wherein the gas laser is any one of an excimer laser, a copper vaporlaser, an argon laser, a He-Ne laser, a CO₂ laser, a He-Cd laser and anitrogen laser.
 14. A microscope system of claim 13, wherein the gaslaser is of a mode-locked type.
 15. A microscope system of claim 9,wherein the solid laser is any one of a Nd:YAG laser, a Ti sapphirelaser, a YLF laser and a ruby laser.
 16. A microscope system of claim15, wherein the solid laser is of a semiconductor-laser-excited type.17. A microscope system of claim 15, wherein the solid laser is of amode-locked type.
 18. A microscope system of claim 9, wherein saidmicroscope body has at least one of a nonlinear media and a wavelengthmodulating element for converting a wavelength of a laser beam from thegas laser, the solid laser or the semiconductor laser.
 19. A microscopesystem of claim 18, wherein the nonlinear media or the wavelengthmodulating element is a nonlinear crystal.
 20. A microscope system ofclaim 18, wherein the nonlinear media or the wavelength modulatingelement is a Raman shifter.
 21. A microscope system of claim 18, whereinthe light having the wavelength λ1 is prepared by modulating awavelength of a fundamental-wave of the gas laser or the solid laserwith the nonlinear media or the wavelength modulating element.
 22. Amicroscope system of claim 18, wherein the light having the wavelengthλ1 is prepared by modulating a wavelength of harmonic-wave of the gaslaser or the solid laser with the nonlinear media or the wavelengthmodulating element.
 23. A microscope system of claim 18, wherein thelight having the wavelength λ2 is prepared by modulating a wavelength ofa fundamental-wave of the gas laser or the solid laser with thenonlinear media or the wavelength modulating element.
 24. A microscopesystem of claim 18, wherein the light having the wavelength λ2 isprepared by modulating a wavelength of a harmonic-wave of the gas laseror the solid laser with the nonlinear media or the wavelength modulatingelement.
 25. A microscope system of claim 9, wherein in a resonator ofthe gas laser, the solid laser or the semiconductor laser, there isprovided at least one of a ring-shaped zonal mirror, a zonal diffractiongrating, a Fresnel zone plate, a zonal aperture, and a phase plate whichgives a phase difference in which electric fields axially symmetric in aplane normal to the optical axis are shifted by π from each other.
 26. Amicroscope system of claim 1, wherein any of a gas laser, a solid laserand a semiconductor laser is provided as said light source for the lighthaving the wavelength λ2.
 27. A microscope system of claim 26, whereinan oscillation wavelength of any of the gas laser, the solid laser andthe semiconductor laser is the wavelength λ2.
 28. A microscope system ofclaim 26, wherein a harmonic-wave of an oscillation wavelength of any ofthe gas laser, the solid laser and the semiconductor laser has thewavelength λ2.
 29. A microscope system of claim 26, wherein a sumfrequency of, or a difference frequency between, an oscillationwavelength of any of the gas laser, the solid laser and thesemiconductor laser and a harmonic-wave of the oscillation wavelengthhas the wavelength λ2.
 30. A microscope system of claim 1, wherein saidcondensing optical system for the light having the wavelength λ2 has aphase plate having a refractive-index distribution or anoptical-path-difference distribution which gives, to the beam obtainedby condensing the light having the wavelength of the λ2, a phasedifference distribution in a plane normal to an optical axis of thebeam.
 31. A microscope system of claim 1, wherein said condensingoptical system for the light having the wavelength λ2 has a zonaloptical system.
 32. A microscope system of claim 1, wherein saidcondensing optical system for the light having the wavelength λ2 has adiffractive optical system.
 33. A microscope system of claim 1, whereinsaid condensing optical system for the light having the wavelength λ2has an axicon.
 34. A microscope system of claim 1, wherein saidmicroscope body has an emission condensing optical system for condensingan emission from the molecule to said emission detector.
 35. Amicroscope system of claim 34, wherein said emission condensing opticalsystem has a sharp cut filter.
 36. A microscope system of claim 34,wherein said emission condensing optical system has a notch filter. 37.A microscope system of claim 34, wherein said emission condensingoptical system has a band-pass filter.
 38. A microscope system of claim37, wherein the band-pass filter transmits the emission from themolecule while not transmitting the light having the wavelength λ1 andthe light having the wavelength λ2.
 39. A microscope system of claim 1,wherein said adjusted specimen is sealed by a seal device made of asubstance transmitting the light having the wavelength λ1 and the lighthaving the wavelength of λ2.
 40. A microscope system of claim 39,wherein the substance is synthetic quartz SiO2, CaF2, NaF, Na3AlF6, LiF,MgF2, SiO2, LaF3, NdF3, Al2O3, CeF3, PbF2, MgO, ThO2, SnO2, La2O3 orSiO.
 41. A microscope system of claim 1, wherein said adjusted specimenis covered by a cover device made of a substance transmitting the lighthaving the wavelength λ1 and the light having the wavelength of λ2. 42.A microscope system of claim 1, what said microscope body has acontinuous-wave laser which is separate from said light sources for thelight having the wavelength λ1 and the light having the wavelength λ2,and wherein a beam obtained by condensing the continuous-wave laser onsaid adjusted specimen has a phase distribution in which the phase isshifted by π at a symmetric position with respect to the optical axis ofthe beam in the plane normal to the optical axis.
 43. A microscopesystem of claim 1, wherein said microscope body has a device forrelatively scanning, on said adjusted specimen, with a beam obtained bycondensing a continuous-wave laser on said adjusted specimen,independently of a beam obtained by condensing the light having thewavelength λ1 and the beam obtained by condensing the light having thewavelength of λ2.
 44. A microscope system comprising: an adjustedspecimen; and a microscope body; wherein said adjusted specimen is dyedwith a molecule which has three electron states including at least aground state; wherein said microscope body includes: a light sourceoperable to provide light having a wavelength λ1 for exciting themolecule from the ground state to the first electron excited state; alight source operable to provide light having a wavelength λ2 forexciting the molecule in the first electron excited state to the secondor higher electron excited state; a condensing optical system operableto condense the light having the wavelength λ1 and the light having thewavelength λ2 on said adjusted specimen; an overlap device operable topartially overlap an irradiation region of the light having thewavelength λ1 and an irradiation region of the light having thewavelength λ2 on said adjusted specimen; and an emission detectoroperable to detect an emission upon deexcitation of the excited moleculeto the ground state; wherein a region of the emission upon deexcitationof the molecule from the first electron excited state to the groundstate is inhibited by irradiating the light having the wavelength λ1 andthe light having the wavelength λ2 through said overlap device; andwherein a beam obtained by condensing the light having the wavelength λ2has a phase distribution in which the phase is shifted by π at asymmetric position with respect to an optical axis of the beam in aplane normal to the optical axis.
 45. A microscope system of claim 44,wherein the beam obtained by condensing the light having the wavelengthλ2 has a phase distribution in which the phase changes continuously from0 to 2πwhen turned once around the optical axis in a plane normal to theoptical was.
 46. A microscope system of claim 44, wherein an excitationwavelength band from the first electron excited state to the secondelectron excited state and an excitation wavelength band from the groundstate to the first electron excited state are different.
 47. Amicroscope system of claim 44, wherein an optical axis of a beamobtained by condensing the light having the wavelength λ1 and theoptical axis of the beam obtained by condensing the light having thewavelength λ2 are coaxial.
 48. A microscope system of claim 44, whereinthe beam obtained by condensing the light having the wavelength λ2 has aphase distribution in which the phase changes discontinuously from 0 to2π when turned once around the optical axis in the plane normal to theoptical axis.
 49. A microscope system of claim 44, wherein the beamobtained by condensing the light having the wavelength λ2 is a Besselbeam.
 50. A microscope system of claim 44, wherein the beam obtained bycondensing the light having the wavelength λ2 is a laser beam having avibrational mode of any one of a Gauss's type, Laguerre's type andHermitian's type.
 51. A microscope system of claim 44, wherein any of agas laser, a solid laser and a semiconductor laser is provided as saidlight source for the light having the wavelength λ1.
 52. A microscopesystem of claim 44, wherein any of a gas laser, a solid laser and asemiconductor laser is provided as said light source for the lighthaving the wavelength λ2.
 53. A microscope system of claim 44, whereinsaid condensing optical system for the light having the wavelength λ2has a phase plate having a refractive-index distribution or anoptical-path-difference distribution which gives, to the beam obtainedby condensing the light having the wavelength of the λ2, a phasedifference distribution in a plane normal to an optical axis of thebeam.
 54. A microscope system of claim 44, wherein said condensingoptical system for the light having the wavelength λ2 has a zonaloptical system.
 55. A microscope system of claim 44, wherein saidcondensing optical system for the light having the wavelength λ2 has adiffractive optical system.
 56. A microscope system of claim 44, whereinsaid condensing optical system for the light having the wavelength λ2has an axicon.
 57. A microscope system of claim 44, wherein saidmicroscope body has an emission condensing optical system for condensingan emission from the molecule to said emission detector.
 58. Amicroscope system of claim 44, wherein said adjusted specimen is sealedby a seal device made of a substance transmitting the light having thewavelength λ1 and the light having the wavelength λ2.
 59. A microscopesystem of claim 44, wherein said adjusted specimen is covered with acover device made of a substance transmitting the light having thewavelength λ1 and the light having the wavelength λ2.
 60. A microscopesystem of claim 44, wherein said microscope body has a continuous-wavelaser which is separate from said light sources for the light having thewavelength λ1 and the light having the wavelength λ2, and wherein a beamobtained by condensing the continuous-wave laser on said adjustedspecimen has a phase distribution in which the phase is shifted by π ata symmetric position with respect to the optical axis of the beam in theplane normal to the optical axis.
 61. A microscope system of claim 44,wherein said microscope body has a device for relatively scanning, onsaid adjusted specimen, with a beam obtained by condensing acontinuous-wave laser on said adjusted specimen, independently of a beamobtained by condensing the light having the wavelength λ1 and the beamobtained by condensing the light having the wavelength λ2.
 62. A methodfor illuminating an adjusted specimen using a microscope body, saidmethod comprising: dying the adjusted specimen with a molecule which hasthree electron states including at least a ground state and which has anexcited wavelength band from a first electron excited state to a secondelectron excited state which overlaps a fluorescent wavelength band upondeexcitation through a fluorescence process from the first electronexcited state to a vibrational level in the ground state; providinglight having a wavelength λ1 for exciting the molecule from the groundstate to the first electron excited state; providing light having awavelength λ2 for exciting the molecule in the first electron excitedstate to the second or higher electron excited state; condensing thelight having the wavelength λ1 and the light having the wavelength λ2 onthe adjusted specimen; partially overlapping an irradiation region ofthe light having the wavelength λ1 and an irradiation region of thelight having the wavelength λ2 on the adjusted specimen; and detectingan emission upon deexcitation of the excited molecule to the groundstate; inhibiting a region of the emission, upon deexcitation of themolecule from the first electron excited state to the ground state, byirradiating the light having the wavelength λ1 and the light having thewavelength λ2; and wherein a beam obtained by condensing the lighthaving the wavelength λ2 has a phase distribution in which the phase isshifted by π a symmetric position with respect to an optical axis of thebeam in a plane normal to the optical axis.
 63. A method forilluminating an adjusted specimen using a microscope body, said methodcomprising: dying the adjusted specimen with a molecule which has threeelectron states including at least a ground state; providing lighthaving a wavelength λ1 for exciting the molecule from the ground stateto the first electron excited state; providing light having a wavelengthλ2 for exciting the molecule in the first electron excited state to thesecond or higher electron excited state; condensing the light having thewavelength λ1 and the light having the wavelength λ2 on the adjustedspecimen; partially overlapping an irradiation region of the lighthaving the wavelength λ1 and an irradiation region of the light havingthe wavelength λ2 on the adjusted specimen; and detecting an emissionupon deexcitation of the excited molecule to the ground state;inhibiting a region of the emission, upon deexcitation of the moleculefrom the first electron excited state to the ground state, byirradiating the light having the wavelength λ1 and the light having thewavelength λ2; and wherein a beam obtained by condensing the lighthaving the wavelength λ2 has a phase distribution in which the phase isshifted by π at a symmetric position with respect to an optical axis ofthe beam in a plane normal to the optical axis.